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Citigroup to repay $20 billion in bailout money

Citigroup Inc. said Monday it is repaying $20 billion in bailout money it received from the Treasury Department, in an effort to reduce government influence over the banking giant. The government will also sell its stake in the company.

The New York-based bank was among the hardest hit by the credit crisis and rising loan defaults and got one of the largest bailouts of any banks during the financial crisis. The government gave it $45 billion in loans and agreed to protect losses on nearly $300 billion in risky investments. Wells Fargo & Co. remains the last national bank that has yet to pay back its bailout money.

Citi is selling $20.5 billion in stock …

Reynolds juror removed over alleged remarks

A juror who had been involved in three other courtroom incidentswas dismissed in former Rep. Mel Reynolds' financial fraud trialWednesday after he was accused of telling a prosecutor, "Keep itbrief."

The African-American juror's removal by U.S. District JudgeCharles R. Norgle Sr., and his replacement with a white malealternate, changed the racial balance of the panel to seven whitesand five blacks.

Robert Loeb and William Hooks, Reynolds' defense attorneys,opposed the juror's removal on the grounds that the man, a CTA busdriver, denied making the comment when he was brought into thecourtroom and questioned by Norgle.At the end of Tuesday's session - the trial's …

Statement from Steve Jobs' family after his death

The family of Apple Inc. co-founder and chairman Steve Jobs issued this statement Wednesday in response to his death:

"Steve died peacefully today surrounded by his family.

"In his public life, Steve was known as a visionary; in his private life, he cherished his family. We are thankful to the many people who have shared their …

Mattel 2Q net income more than doubles

Mattel Inc.'s second-quarter net income more than doubled, boosted by sales of Barbie and toys tied into the Pixar smash "Toy Story 3," the toymaker said Friday.

The maker of Barbie and Hot Wheels says net income rose to $51.6 million, or 14 cents per share, from $21.5 million, or 6 cents per share last year. That just missed analyst expectations for net income of 15 cents per share, according to a poll by Thomson Reuters.

Mattel's revenue rose 13 percent to $1.02 billion, matching analyst expectations. A year ago revenue was $898.2 million.

CEO Robert A. Eckert said sales of Barbie and Hot Wheels were strong and said toys tied into …

Shrinkage stabilizing: ; After 50 years of declines, county enrollment about half that in 69

Over the past 50 years, the Kanawha County school system has lostmore than 25,000 students - or nearly as many students as it hascurrently.

As factories and plants closed and downsized, the county'sschools shrunk, vanished or consolidated.

In the past half-century, 142 Kanawha elementaries have closed.

Much of the student population loss comes from job and populationdeclines in the Kanawha Valley. In the 1950's, the county wasgrowing, adding 13,000 people through the decade to begin 1960 with253,000 residents, according to the national Census Bureau. Butthings have contracted since then. In 2007, the county had about191,000 residents.

Over the past few …

Iron will

Athlete won't let diabetes slow him down

Jim Molyneux is like a real-life Superman. That's not only because the 45-- year-old CA from Inglewood, Ont., recently completed his third Ironman competition in as many years. (He swam 3.9 km across Okanagan Lake in Penticton, BC, cycled 180 km, then ditched the bike and ran a full marathon, all in 12 hours, 38 minutes - his best time yet.) Nor is it just that he learned to swim properly only five years ago, or that he often cycles the 56 km to and from the Toronto CA firm where he is a partner. It's because he does all this and more while living with diabetes.

"Nothing is going to stop me from keeping fit," says the 6'6", 217-lb. …

Greek interior minister survives despite riots

Embattled Prime Minister Costas Karamanlis has reshuffled his Cabinet, replacing his finance minister but retaining the interior minister despite criticism of the government's handling of massive riots last month.

Government spokesman Evangelos Antonaros said Finance Minister Giorgos Alogoskoufis will be replaced by his deputy, Yiannis Papathanasiou.

…

Rookie catchers set to make their marks

The law of supply and demand operates as efficiently in baseballas it does in other fields of economic activity.

In 1986, the demand peaked for young outfielders and anexceptional supply of gifted rookies met the challenge. The farmsystems churned out the richest crop of outfielders in two decades.

Leading the harvest was Jose Canseco, who was the AmericanLeague Rookie of the Year after hitting 33 home runs and driving in117 runs while batting .240. Another dozen rookie outfieldersdistinguished themselves, most notably Pete Incaviglia, Ruben Sierra,Danny Tartabull, Cory Snyder, Kevin Mitchell, Barry Bonds, John Krukand John Cangelosi.

Not since 1964 had …

Pakistan Election Fuels Voter Cynicism

ISLAMABAD, Pakistan - A court must still rule whether Gen. Pervez Musharraf's landslide win in Saturday's presidential election is valid, but one loser was clear before the voting even began: public faith in Pakistani politics.

Opposition boycotts, legal wrangling and the efforts of former leaders Benazir Bhutto and Nawaz Sharif to mount a comeback are the stuff of media debate in Pakistan.

But many ordinary Pakistanis feel that power is the preserve of a corrupt elite, too busy securing their own interests to tackle issues such as rising Islamic militancy and the poverty and inflation that feed it.

"All politicians are the same," said Ali Ahmad, a 28-year-old …

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New Keynesians' Theory Hits Home

Suppose for a moment you bought a house in 1989 for $125,000,approximately the average price of a home in the Chicago area.

Five years later, you want to unload. You list the house at asales price of $150,000 but are told that because of a glut in themarketplace comparable homes are selling for only $140,000.

You aren't persuaded and refuse to drop your price. You areconvinced that your house is different in location, decor andamenities from the rest. And maybe, just maybe, a buyer will comealong who is in a hurry to close or who loves the kitchen.

You opt to hold and allow the house to sit idle. So, too, dothousands of other people in the local housing …

Murdoch tabloid spied on hacking victims' lawyers

LONDON (AP) — Rupert Murdoch's British newspaper company spied on two lawyers who represent alleged victims of phone hacking by its tabloid News of the World, the firm acknowledged Monday.

News International said lawyers Mark Lewis and Charlotte Harris "were subject to surveillance," a practice it called "inappropriate."

The BBC and The Guardian newspaper reported Monday that Lewis and Harris were followed and filmed last year by private investigator Derek Webb, who was hired by the now-defunct tabloid to gather evidence in a bid to discredit them.

Webb — who ran a company called Silent Shadow — told the BBC that the surveillance began in early 2010 and included …

Hockey is hot item with local sports enthusiasts

Hockey has traditionally been a sport popular in New England and other regions where temperatures sink well below zero, but players have been going at it in warmer climates over the past two years, according to a floor salesman at Play It Again Sports, which has stores at Harrisburg's Paxton Square Shopping Center and the Capital City Plaza.

"Hockey was a rule way up in the Northeast, but now hockey is the biggest recognized sport around here," said Tom, the salesman who would only give his first name. No matter whether it's street, ice or in-line hockey, he said, "More people are playing it than ever before." And yes, he said, this includes puckers who are female.

Overall, revenues for sporting goods stores are growing, according to the National Sporting Goods Association, which is the trade group for those retailers.

In 1992, the sporting goods industry recorded total revenues of $34.8 billion. Revenues were $41.6 billion in 1996 and are projeced to be $42.8 billion this year, according to NSGA.

Based on NSGA's 1996 survey, snowboarding is the fastest-growing sport, scoring a 32.5 percent increase over 1995 figures with a total of 3.7 million participants. Soccer was in the number two spot with a 15.6 percent increase over 1995 figures. However, the number of soccer participants is 13.9 million. Target shooting, softball and kayaking/rafting top out the five fastest-growing sports on the survey.

There is a growing interest in archery also. Michael Eitland, Ferguson's Sports products manager, said bow and arrow hunting is popular now compared to rifle use. "It's hard to tell just yet, but it seems that more people are replacing rifle hunting with bow hunting. Like with the difference in fly-fishing and regular fishing, you might say that archery hunting is more specialized than rifle hunting," he said. Eitland, whose store is on Mountain Road in Harrisburg, said just how popular bows and arrows are with hunters will be clearer in a few weeks. Oct. 4 marked the first day of the open archery season and rifle hunters begin their sport the day after Thanksgiving.

Archers are growing in numbers, but hunting overall, said Eitland, is not quite as popular as it once was. "We are seeing fewer younger people interested in hunting, because it's sort of a generational thing. If the parent's don't hunt, it's not likely these days that their children will want to hunt."

Around Central Pennsylvania, the big game is still deer. And while Eitland said bows and rifles both do the job, archery hunting may be considered safer because closer distances to the target are required. Still, archery hunting requires more stringent practice, and old-time hunters prefer rifles.

On fishing, Eitland noted that some aspects of the sport are not declining as swiftly as others, but overall "general fishing has been dropping off for 10 years. On the other hand, fly-fishing climbed steadily for a few years, then began to drop. Also, fishing licensing has been on the decline for the past 10 years, although this varies from area to area, and you really can't count on where will be up or down. Around here, it's sort of steady in season."

While some people take their sports seriously, some treat sporting activities like a trend, Eitland said. "Remember that Robert Redford movie, 'A River Runs Through It?' Well, after that came out, a lot of people wanted to fly-fish. It caused quite a stir with the Yuppie crowds who thought it was cool, so they got into it for a little while."

Speaking of what' s cool, there's always clothing gear to go along with the sport. Dan Simmons sells sports clothing at Kelly's Sports Express in the Colonial Park Mall, Harrisburg. The big items at his store are the footwear, and that's what is attracting those between the ages of 16 and 25, said Simmons. And if you've wondered if there's any end in sight to the constantly skyrocketing prices for a pair of Air Jordans and other Nike Products, Reeboks, Avias or Filas, apparently there is no cost ceiling on the horizon.

"I've listened to more than a few it heated arguments in the store, parent's not wanting to pay the prices that keep going up for these shoes," he said. Still, Simmons insists that although the parents can't justify spending as much as $150 to $200 for a pair of athletic shoes, "they end up buying them anyway. I hear lots of complaints, but the shoes keep selling." And athletic sneakers costing in the hundreds of dollars certainly require being offset with a 'pricey' sporting outfit to complete the ensemble. "Once they get the shoes, the parents are pushed (by kids) to buy running suits, T-shirts, sweatshirts, whatever matches the shoes," Simmons said.

And hearing the arguments isn't easy for Simmons, especially when he further daunts parents by advising them to purchase shoes that actually fit, rather than trying to buy shoes "that the child can grow into. I have to advise against that, because the shoe has to fit, or it can be dangerous for the wearer," he said. "We get new styles and makes of shoes in every week. And depending on what the shoe will be used for -- a game, running, hiking -- it needs to be styled specifically for that (activity)," Simmons said.

With the advent of the 1997 Women's National Basketball Association, girls are heating up cash registers, wanting shoes and gear worn by female star basketball players. But so far, Simmons' store has had to miss out on that business. "Lady Footlocker has an exclusive on the WNBA items until next year, and I'm sure other sports stores will start getting it in when '98 rolls around," he said.

Football, basketball, snow-skiing and track sports are four staple sports here to stay said Tom, at Play It Again Sports. He believes that "products are being built with more quality, and for some sports, the equipment is being modified to improve the player's game." For instance, golf clubs, the irons and the woods, are available with larger heads, and they're getting more popular. This helps learners, and it's just good for the game. And since Tiger Woods, we're getting a lot more young people interested in playing golf and they want those heads," said Tom.

As for what's new and what's trendy, Tom said he thinks the alternative sport of aggressive in-line skating, which involves skating down steps, zooming off rails and combining acrobatics with skating, appeared trendy at first, "but it's not really a trend. It's a specialty sport that younger people are doing. I think it'll stick around, because the skaters are getting more creative."

Our first survey of PSA systems

A review of new software products for your business

For years, popular vendors of accounting and ERP systems neglected the needs of professional services organizations such as accounting, consulting, architectural and engineering firms. Yet these organizations' needs are very different from those of companies that distribute or manufacture products. Fortunately, vendors have now awakened to this fact and have come out with a variety of professional service automation (PSA) systems. That is why we arc taking a tour of the landscape with our first annual survey of PSA products.

Although geared specifically to professional servicebased organizations, PSA can be used by any organization that manages projects and tracks time, such as internal IT or R&D departments. The Aberdeen Group, one of the leading information technology analysts, describes it this way: "PSA supports the core business processes of services-centric organizations to more efficiently utilize people and streamline the project lifecycle to save time, cut costs and increase revenues." PSA systems typically cover most, if not all, of the core business processes for professional service organizations. But there are other systems such as timesheets, time billing and practice management that contain components of PSA and which we have included under the PSA umbrella.

Apart from the vendors that have introduced PSA software products, many accounting system vendors are now offering their own solutions. And then there are those that have targeted a narrow range of professional services such as accounting.

In the accompanying survey on our website, we have tried to sort out all the choices. The products run the gamut from simple to sophisticated. At one end are timesheet systems, which provide timesheets, expense reporting and project management. Next come timebilling systems that include billing and sometimes purchasing, allowing for accumulation of external costs for products and services. When targeted to accountants or lawyers, these time-billing systems are usually called practice management systems.

At the other end of the spectrum are integrated solutions that are meant to meet all the system needs of professional service organizations - timesheet, time billing, customer relationship management, opportunity management, knowledge management and resource management. (For an explanation of the functionality associated with PSA, see "Answering the call," November 2002, at www.CAmagazine.com.) General ledger, accounts receivable and accounts payable are often not included with PSA, but there are links with many of the leading accounting systems.

Some professional service organizations that lack the internal infrastructure to support a PSA system rent their solutions from application service providers. The ASP hosts the application on its Internet site, which is typically equipped with state-of-the-art technology and security, thereby allowing you to avoid the costs of managing the computer and database that goes with it. Some organi/ations worry about security but their own systems usually don't come close to the high level of security demanded of an ASP. That's why an ASP seems like a good choice for many professional service organizations that lack technical resources. Do you think it's just a coincidence that ASP is PSA spelled backward?

The vendors of only 25 products met the deadline for this survey. More have asked to be added to the list, so we will update the chart in a couple of months. In the meantime, we hope the survey makes some sense of the wide world of PSA.

For an expanded version of this article, go to www. CAmagazine.com/PSAsurveyl.

[Author Affiliation]

Michael Burns, MSiA, CA, is president oj 180 Systems (http://www.180systems.com), which provides independent consulting advice in the selection and implementation of business systems. Michael can be reached at 416963-1296 or by email at mburns@180systems.com

Force Microscopy of Nonadherent Cells: A Comparison of Leukemia Cell Deformability

ABSTRACT

Atomic force microscopy (AFM) has become an important tool for quantifying mechanical properties of biological materials ranging from single molecules to cells and tissues. Current AFM techniques for measuring elastic and viscoelastic properties of whole cells are based on indentation of cells firmly adhered to a substrate, but these techniques are not appropriate for probing nonadherent cells, such as passive human leukocytes, due to a lateral instability of the cells under load. Here we present a method for characterizing nonadherent cells with AFM by mechanically immobilizing them in microfabricated wells. We apply this technique to compare the deformability of human myeloid and lymphoid leukemia cells and neutrophils at low deformation rates, and we find that the cells are well described by an elastic model based on Hertzian mechanics. Myeloid (HL60) cells were measured to be a factor of 18 times stiffer than lymphoid (Jurkat) cells and six times stiffer than human neutrophils on average (E^sub ∞^ = 855 � 670 Pa for HL60 cells, E^sub ∞^ = 48 � 35 Pa for Jurkat cells, E^sub ∞^ = 156 � 87 for neutrophils, mean � SD). This work demonstrates a simple method for extending AFM mechanical property measurements to nonadherent ? cells and characterizes properties of human leukemia cells that may contribute to leukostasis, a complication associated with acute leukemia.

INTRODUCTION

In some diseases, the mechanical properties of individual cells are altered. For example, osteoarthritic chondrocytes (cartilage-producing cells) have been shown to be less stiff than normal chondrocytes, and malignant hepatocytes (liver cells) have been shown to be stiffer than normal hepatocytes (1-3). In diseases of the blood, changes in cell mechanical properties can have profound effects on the cells' ability to flow normally through the vasculature, since increased stiffness impedes progress of cells through small capillaries (4). The reduced deformability of erythrocytes infected with Plasmodium falciparum, the parasite that causes malaria, has been shown to hinder the cells' ability to flow through microfabricated channels (5). Recent research on diabetes mellitus suggests that some complications of the condition can be attributed to increased lymphocyte stiffness (6). In acute leukemia, immature blood cells of the myeloid or lymphoid lineages, called myeloblasts and lymphoblasts, respectively, proliferate uncontrollably. Decreased deformability of these cells, as well as increased adhesion and transmigration, is thought to be linked to leukostasis, a poorly understood condition in which cells aggregate in the vasculature (7). This condition often results in intracranial hemorrhage and respiratory failure that rapidly leads to death, and current therapies based on removal of leukemia cells from the circulation have not proven to decrease mortality (8). Better knowledge of biophysical changes in leukemia cells such as deformability is necessary for improved understanding of the disease, but no widely accepted method or model exists for quantifying the mechanical properties of leukemia cells relevant to leukostasis.

Atomic force microscopy (AFM), first developed as a surface imaging tool (9), can also be used to measure the stiffness of cells firmly adhered to a substrate (10). The primary method of measuring stiffness is indenting the cell with a flexible cantilever driven at a constant extension rate (piezo extension rate) with respect to the sample. The deflection of the cantilever as it indents the cell, which is linearly related to loading force for small deflections, is recorded by reflecting a laser off the cantilever into a split photodiode. A linear elastic model of the cell based on Hertzian mechanics (11) is commonly used with AFM deflection data to determine cell elasticity (12). In addition to constant piezo extension rate measurements, elastic and viscoelastic properties of cells can be obtained from creep experiments (13) and oscillating indentations (14,15) based on similar elastic models.

Nonadhesive cells, such as normal and malignant leukocytes before activation of the inflammatory response, pose a challenge for AFM because they tend to slip from under the cantilever tip under an applied load. Although nonadhesive cells can occasionally be probed using AFM without slippage (13), the experimental throughput is low and may favor those cells which have some level of adherence to the surface. One solution is to coat the substrate surface with poly-peptides, making it sticky to the cell. Attaching a nonadherent cell to a surface coated with fibronectin or poly-lysine will prohibit it from moving while probed but can change its morphology and mechanical properties, similar to the ? changes in cell properties seen upon neutrophil activation (16). Nonadherent cells trapped within Millipore filters have been used for AFM imaging (17), but the pressure used to trap the cells is likely to significantly deform the cells and may change their elastic response.

Micropipette aspiration has been used extensively to determine mechanical properties of fully differentiated leukocytes such as neutrophils (18-23). During micropipette aspiration, a section of membrane and cytoplasm of the cell are drawn into the pipette by a pressure differential. This technique can be used to describe an apparent membrane tension and cytoplasmic viscosity using the liquid droplet model, in which the cell is assumed to be a viscous fluid-filled bag with a constant surface tension. Since aspiration of a cell draws in cytoplasm more readily than nucleus, application of this technique to leukemia cells may overestimate their deformability due to a characteristically high nucleus/cytoplasm ratio (24). Whole cell aspiration is potentially more appropriate for leukemia cell deformation and has been used to describe HL60 mechanical behavior (25,26). However, previous analysis makes use of a power-law fluid model, which is not able to describe a static deformation such as when a cell plugs a capillary, as is believed to occur in leukostasis. A simple method for characterizing and comparing the deformability of leukemia cells at low deformation rates is needed.

We used microfabricated wells (Fig. 1) to mechanically immobilize and study the deformability of HL60 and Jurkat cells, prominent acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) cell lines, and human neutrophils. AML has been associated with leukostasis at a significantly lower cell concentration than ALL, but the reason for this clinical observation is not known. Comparative measurements of deformability will help in understanding the pathophysiology of the disease. We analyzed our experimental data with two models-a Hertzian mechanics and a liquid droplet model-and determined that the Hertz model is a more appropriate description of these cell lines at low deformation rates. HL60s were found to be significantly stiffer than Jurkat cells and neutrophils, consistent with a model of leukostasis in which stiffness contributes to vessel blockage.

MATERIALS AND METHODS

Cell culture

In this study, the HL60 and Jurkat cell lines (American Type Culture Collection, Manassas, VA, ATCC numbers TIB-152 and CCL-240, respectively) were used as models for AML and ALL. respectively. Cells were cultured in RPMI 1640 containing 10% fetal bovine serum (Gibco, Carlsbad, CA) and maintained in a 5% CO2 humidified atmosphere at 37�C. Before force microscopy measurements were made, cells were incubated in RPMI 1640 without fetal bovine serum for 24 h at 37�C and 5% CO2 to synchronize cells in the G0 phase of the cell cycle (27,28).

To isolate neutrophils, whole blood was drawn from healthy donors and collected in heparin. Histopaque 1077 (Sigma-Aldrich, St. Louis, MO) was layered atop Histopaque 1119 to create a dual density gradient. Whole blood was then layered atop the upper gradient and the solution was centrifuged at 700 � g for 30 min, which isolated the neutrophil layer between the two histopaque layers. All layers above the neutrophils were discarded, and the neutrophils were collected and pelleted. The cell pellet was then resuspended in sterile distilled water to lyse any red blood cell contaminanls within the solution. After 10 s, 1 part of 10� Hanks buffered salt solution was added. Cells were pelleted and resuspended twice in RPMI to eliminate red blood cell ghosts.

For cell diameter and nucleus/cytoplasm ratio measurements, a Zeiss Axiovert 200 microscope (Carl Zeiss, Thomwood, NY) with a Zeiss 100 � 1.3 numerical aperture (NA) oil immersion phase objective was used. Nuclei were fluorescently stained with Hoechst 33342 (Molecular Probes, Eugene, OR) for visualization, and cell morphology was imaged with phase microscopy. Metamorph software (Molecular Devices, Downingtown, PA) was used to quantify the cross-sectional areas of the nuclei and whole cells.

HL60, Jurkat, and neutrophils cells were labeled with Alexa Fluor 546 phalloidin (Molecular Probes) following the standard protocol to image the actin cytoskeleton. Images were taken with a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss).

Microwells

To create microfabricated wells, or microwells, for mechanical immobilization of the cells (Fig. 1), the photocurable epoxy SU-8 5 (Microchem, Newton, MA) was spun onto piranha-cleaned Borofloat glass wafers (Precision Glass and Optics, Santa Ana, CA) and prebaked (2 min at 65�C and 5 min at 95�C). The wafer was then exposed through a mask with a mercury arc lamp (175 mJ/cm^sup 2^), postexposure baked (1 min at 65�C and 2 min at 95�C), and developed in SU-8 developer (Microchem) for 3 min. Well depth was controlled by spin speed during SU-8 application and measured to be 11.0 �m with an Alpha-Step IQ Surface Profiler (KLA-Tencor, San Jose, CA). Wells were patterned in arrays with diameters ranging from 8 to 20 �m, though for the experiments in this work microwells with diameters of 13.6 � 0.3 �m (mean � SD, n = 10) were used for HL60 and Jurkat indentation and microwells with diameters of 10.8 �m � 0.6 �m (mean � SD, n = 18) were used for neutrophil indentation. No differences in cell morphology or viability were observed between cells incubated on SU-8 and glass surfaces.

AFM experiments

All force microscopy measurements were obtained on a modified commercial AFM. A Bioscope AFM (Veeco, Santa Barbara, CA) mounted onto a Zeiss Axiovert 25 held the fluid-cell-mounted cantilever (Microlevers, Veeco). For deformability measurements, we used a closed-loop single-axis 50 �m range, 0.7 nm accuracy piezoelectric positioning platform ("piezo", Mad City Labs, Madison, WI) instead of the piezoelectric tube on the Bioscope AFM head. The piezo platform and photodiode signal were controlled by an RHK SPM 7 controller and RHK SPM32 software (RHK Technology, Troy, MI). V-shaped gold-coated silicon nitride cantilevers (Veeco) with a spring constant of 9-11 pN/nm (calibrated by the thermal noise method (29)) were used in all experiments.

Cells were pipetted onto the wafer and allowed to settle into the microwells. Some cells were moved into wells for force microscopy by gently herding them with the cantilever tip. This movement did not cause any measurable change in cell modulus when analyzed with a two-way ANOVA with cell-type (HL60 versus Jurkat) and microwell status (cells moved into microwells versus cells already in microwells) as the independent variables and cell stiffness as the dependent variable (n^sub HL60^ = 15. n^sub Jurkat^ = 16). Cells pushed into the microwells with the AFM cantilever and cells that had fallen into the microwells on their own did not differ in stiffness (p = 0.93). Also, there was no cell-type versus microwell status interaction (p = 0.63), indicating that the difference in stiffness between HL60s and Jurkats was the same for cells that were moved into the microwells as for cells that had fallen into the microwells.

All data were taken at 25�C. Though temperature likely affects the material properties of the cells studied (30), this study is primarily comparative. Experiments were performed within 1 h after cells were removed from the incubator. HL60 apparent stiffness increased slightly over that 1 h period (R^sup 2^ = 0.10, p = 0.01). Jurkat and neutrophil apparent stiffness showed no significant increase over time (R^sup 2^ < 0.001, p = 0.92 and R^sup 2^ = 0.06, p = 0.24, respectively), indicating that the null hypothesis of no correlation cannot be rejected.

A microwell-trapped cell was moved underneath the cantilever tip with a two-axis translation stage. Mechanical properties were determined by extending the piezo platform at a constant rate, deflecting the cantilever upon contact with the cell until ~800 pN of force was applied or the cell was indented 3 �m (~25% of cell diameter). Substrate effects may influence stiffness values at indentations more than 10% of cell diameter for the Hertz model (31), though we observed no deviation in the model fit over the entire indentation range. Furthermore, substrate effects would not be expected to affect comparative studies of leukemia cell properties. Only the loading curve was used in analysis of the data.

To determine the role of deformation rate and cell viscosity on deformation response, the piezo extension rate was varied between 24 nm/s and 8643 nm/s. The viscosity of the media had negligible effects on cantilever deflection during probing. This was determined by acquiring indentation data on a hard glass surface at all rates and finding no difference in the loading curves. The cells were probed in a random order of rates to avoid measurement bias. A population of HL60 (n = 60), Jurkat (n = 37), and neutrophil (n = 26) cells was indented to determine the average elasticity of the cells in the limit of low deformation rates (apparent equilibrium Young's modulus, E^sub ∞^). For this experiment, the platform moved at 415 nm/s and cells were indented five times each. Student's t-test was used to determine if deformability of the cell types statistically differed.

Experiments were conducted with both the pyramid AFM tip and a 10-�m-diameter sphere indenter. The pyramid silicon nitride tip is 3 �m in height with a 35� half-angle. To attach a sphere to the cantilever tip, cantilever chips were mounted on a manual three-axis micromanipulator and visualized with light microscopy. The cantilever of interest was lowered onto a glass slide containing 5-min epoxy (Devcon, Danvers, MA), and the end of the cantilever was wet with the adhesive. The cantilever tip was then lowered onto a glass slide with dried 10-�m polystyrene beads and adhered to an individual bead. Bonding between the cantilever and the bead was confirmed with light microscopy.

Modeling and analysis

Numerous mechanical models have been used to characterize cell deformability, including the liquid droplet (19), Hertzian mechanics (linear elastic) (10), Maxwell fluid with constant surface tension (18), standard linear solid (32), power-law fluid (26), compound drop (33), and variations of these (34,35). In this work we consider two representative models in the limit of low deformation rate: the liquid droplet and Hertzian mechanics models. The Hertzian mechanics model was selected because it accounts for probe geometry and has been extensively used with AFM (10,12,36). Furthermore, at low deformation rates, the standard linear solid model reduces to a linear elastic model. The liquid droplet model was selected because of its extensive use with leukocytes (37). For low deformation rates, the Maxwell fluid with constant surface tension model reduces to the liquid droplet model.

Liquid droplet model

The liquid droplet model with a constant cortical tension has been used extensively to describe neutrophils (19,37). This model has been used predominantly with micropipette experiments where membrane tension is determined by aspirating part of the cell into the pipette. Lomakina et al. (40) derived the liquid droplet model for the indentation of a cell with a sphere, and we extend the derivation for a pyramid indenter appropriate for our AFM measurements.

The liquid droplet model described here rests on several assumptions: i), the internal contents of the cell are a homogeneous viscous liquid, ii), the cortical tension is constant around the cell, iii), the indenter is moving at a rate slow enough so the viscosity of the contents and the membrane are inconsequential, iv), the cortical shell conformally deforms around the tip during indentation, and v), the radius of the cell remains constant during indentation. The validity of these assumptions is examined in the Discussion section. A custom Igor script was written to determine cortical tension and contact point using a nonlinear least squares optimization method. Recently, Sen et al. (41) developed a theoretical model for indenting a red blood cell adhered to a surface with a cone-shaped AFM tip. This model could be modified for a spherical cell not adhered to a surface to avoid some of the assumptions used here, but there is no simple analytical solution for it.

RESULTS

We considered three questions. First, at what deformation rates are any viscous contributions from the cytoplasm to the apparent cell elasticity minimized? Second, which model (Hertzian mechanics versus liquid droplet) more accurately describes the deformability of the cells? Third, how different are the mechanical properties of leukemia cells from myeloid and lymphoid lines when compared to each other and to normal neutrophils? Answers to the first two questions are described here in detail for the myeloid (HL60) cell line but were also obtained for the lymphoid (Jurkat) cell line and neutrophils (see Table 1 for a summary of these data, and Supplementary Material for more details on these data). The three cell types are quantitatively compared to answer the third question and provide biophysical insight into clinical complications associated with acute leukemia.

Cell deformability measurements

A sample curve from an indentation experiment into a HL60 cell is shown in Fig. 3. When the piezo position is positive, the cantilever tip has not yet come into contact with the cell. As the tip contacts the cell, the cantilever begins to deflect and the curve increases nonlinearly. The influence of deformation rate, and hence cell viscosity, on mechanical property measurements obtained with the AFM was evaluated by conducting experiments at piezo extension rates from 24 to 8643 nm/s. An examination of one specific HL60 cell shows a viscous response at increasing piezo extension rates, since deflection curves increase in slope as rate increases (Fig. 4 A). This trend is seen clearly when using a Hertzian mechanics model to determine apparent stiffness (Fig. 4 B), Apparent stiffness remains relatively constant at and below 415 nm/s but increases monotonically at higher piezo extension rates. The constant apparent stiffness at low deformation rates is seen more clearly in Fig. 4 C, where HL60 cells show no increase in apparent stiffness when piezo extension rate is increased from 24 to 415 nm/s (n = 8), indicating that deformability measurements in this range of piezo extension rates are not significantly influenced by viscosity. For this analysis, apparent stiffness for each cell within the sample was normalized due to the variance of stiffness across cells. This domain of low deformation rates, in which we denote the apparent stiffness E^sub ∞^, simplifies the models to which the experimental data are compared and are likely most relevant for the case of a cell plugging a capillary in leukostasis.

This same viscous response at higher piezo extension rates was seen in an identical rate analysis performed on Jurkat cells and neutrophils. Both cell types had similar plateaus of apparent stiffness at low piezo extension rates with apparent stiffness increasing monotonically with increasing piezo extension rate. The apparent stiffness of neutrophils was level at 501 nm/s and below, whereas that for Jurkat cells was level at 948 nm/s and below. (Please refer to the Supplementary Material for more details of these experiments.)

The microwell walls are not expected to constrain cells during mechanical property measurements, due to size differences between the well and cell diameter. HL60 cell diameter was 12.4 � 1.2 �m (n = 51) and Jurkat cell diameter was 11.5 � 1.5 �m (n = 44), whereas microwell diameter was 13.6 � 0.3 �m (n = 10). Smaller wells (10.8 � 0.6 �m, n = 18) held the similarly smaller neutrophils (8.3 � 0.6 �m, n = 49) for indentation experiments. Cells were selected to be smaller than the wells in which they fell or were placed. For a sample of Jurkat and HL60 cells (n^sub HL60^ = 13, n^sub Jurkat^ = 8), there was a near significant correlation between cell diameter and modulus (R^sup 2^ = 0.16, p = 0.06). However, this rinding should be interpreted in light of the demonstrated effect of cell type on stiffness. HL60 cells, which are shown to be significantly suffer than Jurkat cells, also in general are larger. A partial correlation between cell size and stiffness, controlling for cell type, revealed no significant relationship (p = 0.83). This indicates that once the hypothesized relationship between cell type and stiffness is accounted for, cell size is no longer related to stiffness.

Comparison of Hertzian mechanics and liquid droplet models

The Hertzian mechanics and liquid droplet models described previously were fit to the experimental indentation curves for each cell type. An example fit for a HL60 cell indentation is seen in Fig. 5 A. Example fits for Jurkat and neutrophil data can be seen in Supplemental Fig. 3 in Supplementary Material. The Hertz model was used to determine apparent stiffness (E^sub ∞^) and contact point (z^sub 0^, d^sub 0^), and the liquid droplet model was fit for membrane tension (T) and the contact point (z^sub 0^, d^sub 0^). For each cell type, the Hertz model had a lower mean squared error (MSE) than the liquid droplet model (Table 1). Paired Student's t-test comparing the MSE showed this difference was statistically significant for each cell type (α = 0.05, p^sub HL60^ < 0.001, p^sub Jurkat^ = 0.01, p^sub neutrophil^ = 0.003). Much of the observed error in the liquid droplet model arose from nonnormally distributed residuals near the contact point, which may be due in part to assumptions of the liquid droplet model used here. Based on these fits, we decided to use the Hertzian mechanics model to compare the stiffness of the different cell types.

Comparison of HL60, Jurkat, and neutrophil cell deformability

Myeloid and lymphoid leukemia cells as well as human neutrophils were indented under low deformation rates to determine E^sub ∞^. Indenting at a piezo rate of 415 nm/s was determined to be in the equilibrium regime, where apparent stiffness is not affected by changes in rate, for all cells indented. We report HL60, Jurkat, and neutrophil cells to have an apparent stiffness of 855 � 670 Pa, 48 � 35 Pa, and 156 � 87 Pa (mean � SD, n = 60, 37, and 26 respectively) (Fig. 6, Table 1). Effect of cell type on stiffness is significant (α = 0.05, p < 0.001) when using a one-way ANOVA analysis. Using Bonferroni t-tests for post hoc pairwise analyses, we found HL60 cells to be significantly suffer than Jurkat and neutrophil cells (p < 0.001) and neutrophil cells to be significantly suffer than Jurkat cells (p < 0.001).

DISCUSSION

Modeling

For the three cell types tested, the Hertz model was found to tit the data significantly better than the liquid droplet model, based on paired t-tests described in Results. The mechanical models used here rest on assumptions that affect the determined parameters for stiffness and tension. We examined how the pyramidal indenter model pushing into a liquid droplet could contribute to the observed inaccuracy. First, the model assumes that the cell deforms conformally around the pyramid during indentation. This assumption might not be correct, given previous data which showed that for aspiration experiments with micropipettes with smaller than 1 �m radius, the simple liquid droplet model was not valid due to the bending modulus of the membrane and cortex (42). To test this hypothesis, we indented HL60 cells with a 10-�m-diameter spherical indenter to reduce the effect of the bending modulus. We tit the Hertzian mechanics model for two spheres in contact and fit the liquid droplet model for a spherical punch to the data (Fig. 5 B) and found that the MSEs of the fits were similar to the pyramid punch (MSE^sub Hertz^ = 1.1 nm^sup 2^ vs. MSE^sub droplet^ = 11.3 nm^sup 2^), indicating that cell deformation around the indenter tip is not the primary reason the liquid droplet model does not fit the data as well as the Hertzian mechanics model.

Second, the liquid droplet model assumes the indenter and cell are in static equilibrium at all points during indentation. The repeatability of indentation curves shown in Fig. 4 A as well as the constant apparent stiffness at slow deformation rates in Fig. 4 C supports this assumption. Time constants reported by Lomakina et al. (40) are also consistent with the assumption of static equilibrium.

Although the Hertzian mechanics model fits well to the data, errors in the absolute value of elasticity are expected when using this model to estimate the apparent stiffness at low deformation rates, E^sub ∞^. The assumption that a cell is homogeneous and isotropic is clearly incorrect-leukocytes, like other cells, have a cytoskeleton, organelles, and nucleus that make them inhomogeneous (43). To quantify the influence of local inhomogeneities on our measurements, we compared the standard deviation of E^sub ∞^ of a population of HL60 cells indented with the 10-�m-diameter sphere versus the pyramid tip. We expected the standard deviation for E^sub ∞^ to be reduced because any inhomogeneities would be averaged out over the larger contact area, and indeed it was. The standard deviation for E^sub ∞^ was reduced from 78% of the mean to 53% of the mean (data not shown). Using such a large indenter, however, caused the semiinfinite solid assumption for Hertzian mechanics to be less valid-a model from Dimitriadis et al. (31) showed increased deviation for the Hertz model as indenter tip radius increases for a sample with finite thickness. For example, when indenting a sample with a modulus of 855 Pa with a 4-�m-diameter tip, the apparent modulus is overestimated by 12%. Fora 10-�m-diameter tip, the apparent modulus is overestimated by 85%. Usage of the sphere indenter requires the contact radius to be no more than 10% of the indenter radius for the results to be reliable (44). However, this is difficult when probing soft leukocytes with commercial AFM cantilevers since the cantilevers do not deflect enough when compared to noise in the system to extract adequate data.

The pyramid tip also has limitations when used with the Hertzian mechanics model if the tip (35� half-angle) creates local strains that exceed the linear-elastic assumption. Dimitriadis et al. (31) found this geometry overestimated Young's modulus when compared to that found with a sphere indenter by 60%. The smaller tip does allow for smaller total strain, reducing the effect of the hard glass surface below the cell, which would tend to overestimate the Young's modulus. Nonideal tip geometry could also create errors in estimation of stiffness, for instance, using a blunter tip (one with 5� larger half-angle), whereas still modeling with the expected 35� tip half-angle would result in an overestimation of the apparent stiffness by 20%. Similarly, using a 5� half-angle sharper tip would result in an underestimation of apparent stiffness by 18%.

All simple analytical models have limitations when used to describe constitutive mechanical behavior of cells. Although the Hertzian mechanics model is no exception, it is able to quantify differences in deformability between cell types in a simple and effective manner, which is of interest in the application of cell mechanics to clinical problems where comparisons among cell types are necessary.

Deformability comparison

Cell deformability is thought to play a role in the pathophysiology of leukostasis, as stiffer cells have a higher tendency to mechanically obstruct the vasculature (8,45). Leukostasis has been found to occur at a much higher frequency in AML than ALL at the same cell concentration (8). Clinicians commonly accept that AML cells are more rigid than ALL cells and normal neutrophils (46,47). However, there is scant data that support this assertion. We found HL60 myeloblasts to have a mean E^sub ∞^ of 855 Pa and Jurkat lymphoblasts to have an E^sub ∞^ of 48 Pa (Fig. 6). This difference is significantly greater than found in filtration experiments, which found ALL cells and AML cells equally deformable (7,30), and implies that leukemic cell deformability may play a larger role in leukostasis than previously thought. As described in Results, Jurkat lymphoblasts and neutrophils were found to be significantly less stiff than HL60 myeloblasts. This is consistent with clinical findings that ALL and chronic myeloid leukemia (a leukemia of more mature myeloid cells) rarely go into leukostasis.

Comparisons of leukemia cell deformability were initially performed in the 1970s. With nucleopore filtration experiments, it was reported that ALL cells were more likely to pass through 8 �m pores than AML cells (30). When accounting for cell diameter, however, these cells were reported to be equally filterable. Micropipette experiments showed that myeloblasts were less deformable than leukemic lymphocytes from patients with chronic, or mature, lymphoid leukemia (7). However, because nonblastic lymphocytes rarely cause leukostasis, these findings are not very revealing. Although our measurements directly compared myeloblast and lymphoblast properties, the cells used were from leukemic cell lines and may not reflect the properties of leukemia cells in vivo. Primary leukemia cell measurements must be performed before any definitive conclusions can be made.

Our data are in the same range for Jurkat E^sub ∞^ determined previously with micropipette aspiration using a standard linear solid model (48). Although E^sub ∞^ of HL60 cells has not been measured previously (to our knowledge), we can compare these measurements to stiffness measurements of other cell types. Wojcikiewicz et al. (49) reported that the 3A9 cell, a T-cell hybridoma, has an elasticity of 1.4 kPa when indented at 5000 nm/s, on the order of our findings. Domke et al. (50) found stiffness of adherent osteoblasts away from the stress fibers to be ~500 Pa.

Our values for deformability of neutrophils are also in the range found previously by others. When using the liquid droplet model, we found neutrophils to have a cortical tension of 48 � 20 pN/�m (mean � SD). This is comparable to the findings from micropipette aspiration experiments of Evans and Yeung (35 pN/�m (19)). Needham and Hochmuth (24 pN/�m (51)), and Tsai et al. (27 pN/�m (23)). Our value is slightly higher than the cortical tension found by Lomakina et al. (16-24 pN/�m) when they collided neutrophils into spheres via fluid flow (40).

Biophysical mechanism for stiffness difference

Nuclei have been documented to be substantially larger in most types of leukemias and leukemia cell lines when compared to normal leukocytes (Fig. 7) (52) and have been previously thought to dominate leukemia cell deformability behavior (7). We found that the HL60 cell nucleus is 49 � 6% (mean � SD, n = 40) of the total cross section, the Jurkat cell nucleus is 55 � 8% (mean � SD, n = 47) of the total cross section, and the neutrophil nucleus is 39 � 11% of the total cross section (mean � SD, n = 49), agreeing with previous measurements for AML, ALL, and neutrophils (43,53). When analyzed with a one-way ANOVA, there was a significant effect of cell type on nucleus/whole cell ratio, with significant differences between all ratios in a Newman-Keuls post hoc pairwise analysis (p^sub HL60-Jurkat^ = 0.002, p^sub HL60-neutrophil^ < 0.001, p^sub Jurkat-neutrophil^ < 0.001). Although nuclei of some cells have been shown to be significantly suffer than the whole cell (54,55), our data show that simply having a larger nucleus does not mean that a cell is stiffer, as Jurkat cells were found to be of similar stiffness to the neutrophils even though their nuclei are significantly larger.

To determine if the actin cortex was substantially thicker or denser in the HL60 cells than in the Jurkat or neutrophil cells, we fluorescently labeled the actin cytoskeleton and imaged the cells with confocal microscopy (Fig. 7). Based on those images, we could not conclude that there were any differences in the actin cortex of the leukemia cells that would lead to the difference in stiffness. This raises the interesting question of what governs the mechanical properties of these diseased cells. Whether the stiffness difference observed in our measurements lies in cytoskeletal filament networks (actin, microtubules, or lamins), cytoplasm, nuclear or cell membranes, or a combination of these remains unclear. AFM is an ideal tool for further work on this question.

CONCLUSIONS

In this work, we developed microwells to immobilize non-adherent cells for force microscopy and applied this technique to myeloid and lymphoid leukemia cell lines as well as neutrophils. We determined that the Hertzian mechanics model fits the data well and yielded an apparent equilibrium stiffness for the HL60 that is 18 times higher than that of Jurkat cells and six times higher than that of neutrophils. These findings are consistent with the finding that myeloid leukemias are involved in leukostasis at a higher rate than lymphoid leukemias. Comparing leukemia cell deformability will improve the understanding of leukostasis in acute leukemia. Further studies will be needed to investigate the role of deformability with other hypothesized factors involved in leukostasis, including adhesion and transmigration.

SUPPLEMENTARY MATERIAL

An online supplement to this article can be found by visiting BJ Online at http://www.biophysj.org.

The authors thank J. Alcaraz, O. Chaudhuri, S. Gupta, M. Mofrad, J. Shaevitz, and other members of the Fletcher Lab for technical assistance, discussions, and careful reading of the manuscript. Microfabrication and electron microscopy were performed in the UC Berkeley Microlab and Electron Microscopy Lab. respectively. Confocal microscopy was done at the UC Berkeley Biological Imaging Facility.

This work was supported by a National Science Foundation Graduate Research Fellowship to M.J.R., a National Institutes of Health Ruth L. Kirschstein National Research Service Award and Hammond Research Fellowship of the National Childhood Cancer Foundation and Children's Oncology Group to W.A.L., and a National Science Foundation CAREER Award and University of California Cancer Research Coordinating Committee grant to D.A.F.

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[Author Affiliation]

Michael J. Rosenbluth,* Wilbur A. Lam,*[dagger] and Daniel A. Fletcher*

* Department of Bioengineering, University of California Berkeley and University of California San Francisco/University of California Berkeley Joint Graduate Group in Bioengineering, Berkeley, California; and [dagger] Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of California San Francisco, San Francisco, California

[Author Affiliation]

Submitted May 26, 2005, and accepted for publication January 4, 2006.

Address reprint requests to Daniel Fletcher, PhD, Dept. of Bioengineering, UC Berkeley, 481 Evans Hall, No. 1762, Berkeley, CA 94720-1762. Tel.: 510-643-5624; Fax: 510-642-5835; E-mail: fletch@berkeley.edu.

� 2006 by the Biophysical Society

0006-3495/06/04/2994/10 $2.00

India's economy at risk ; Misgovernance, slowing investments and rising capital costs threaten the India decade. Unless New Delhi acts decisively, sustaining the economic growth rate will be a big challenge.

When you have spent decades in the truck-making business, youhave a sense of what is coming around the corner. For that reasonalone perhaps it is instructive to listen to Managing Director R.Seshasayee's troubles in running a new factory in Pantnagar,Uttarakhand.

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All these troubles are yet to add up to a trend reflecting in theheadline numbers, but the Indian economy looks like it is headedinto rough waters, if not already there. Moderation is alreadyvisible in the growth of industrial output, which hit an 18-monthlow of 2.7 per cent in November, the latest period for which data isavailable. The pressures of runaway food inflation are fast spillingover into the manufacturing sector. Input costs are rising. The soleweapon in use in the fight against inflation - quite inappropriatein the current situation of supply bottlenecks - remains theinterest rate hikes by the Reserve Bank of India, or RBI, which inturn are resulting in a rise in the costs of borrowing.

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There are also new risks looming large from global developmentsthat the government has no control over: rising commodity prices,especially of food and crude oil, for instance. Global oil pricesare ruling above $90 per barrel and will only march northwards asthe recovery in advanced economies strengthens. The government'sfuel and food subsidy bill is mounting. The Food Price Index of theFood and Agriculture Organization of the United Nations hit a recordhigh in December.

Countries like China, Vietnam and Brazil all face double-digitfood inflation. India is not a major food importer yet, but highglobal prices can af fect price sentiment here. "International foodprices have surged sharply this year and the floods in Australia arelikely to exert an upward pressure on grain prices," says M. GovindaRao, Director, National Institute of Public Finance and Policy, aNew Delhi-based think tank. "Increasing commodity prices are a majorthreat to growth."

In short, the overall outlook for the economy is less optimisticthan it was just three months ago. The central bank, known to chooseits words carefully, flagged the heightened risks in its latestpolicy statement on January 25. It expects the rate of growth toslow down. It will announce by how much on May 2, 2011. (Seeinterview 'The Growth Rate May Come Down'). "The combined risks frominflation, the current account deficit and fiscal situationcontribute to an increase in uncertainty about economic stabilitythat consumers and investors will have to deal with," the RBIwarned. "To the extent that this deters consumption and investmentdecisions, growth may be impacted Slower growth can also have asignificant impact on capital inflows, asset prices and fiscalconsolidation," it said.

It worries A. Subba Rao, Group Chief Financial Officer, GMRGroup, that there is no effort from the government to triggergrowth. "Inflow of capital is going to be scarce. Capex will go downand the negative sentiments will affect consumption too. Corporateswill find it difficult to raise capital this year. Such a situationwill last for the whole of 2011-12," he fears. That could make for aweak footing to start the Twelfth Five Year Plan, 2013-17, on - aPlan that will count on half of the Rs 40-trillion total investmentsin the period to come from the private sector.

Subba Rao's prediction of weak capital expenditure in the nextfiscal year stems from the seven interest rate hikes by the RBI overthe past 12 months. The latest increase of 25 basis points in therate at which the central bank finances banks had the CII cryingfoul. "RBI is setting the stage for a series of rate hikes that willhave a negative impact on the investment momentum," Director GeneralChandrajit Banerjee reacted in an unusually sharply wordedstatement, referring to the slowdown in investments.

If GMR, CII and others in corporate India are so down on India,how come the government is unruffled? New Delhi, panned recently byleading businessmen and lawyers for what they called "a governancedeficit", is optimistic that the growth story is intact. Part ofthis is because the government is faced with - or chooses to dealwith - data that comes with a lag of six weeks to three months. Ifit looked around for some forward-looking cues, it might not be socomfortable.

The latest quarterly industrial outlook survey conducted by theRBI in October-December indicates a marginal moderation in overallbusiness expectations for the January-March quarter. HSBC PurchasingManagers' Index, too, shows some moderation in the pace ofmanufacturing sector expansion in December. And, the Indian equitymarkets have shed 10.5 per cent in value in January.

Ahead of a year with five state elections , when FinanceMinister Pranab Mukherjee presents the 2011-12 Union Budget inParliament on February 28, there will be few signs that a robusteconomy is turning. He is likely to be within the 5.5 per centfiscal deficit target he had set for the government.

Ajit Gulabchand

Chairman and Managing Director, HCC

We are losing Rs 2 crore a day due to the abrupt closure slappedby the Ministry of Enviroment & Forests on our Lavasa project. Indiashould have environment standards in place rather than arbitraryaction against projects.Impact: A McKinsey study estimates that landacquisition and environment clearances-related time/cost over-runsfor projects average ~25%.But, as the Economic Advisory Council tothe Prime Minister and the RBI have warned, the deficit target willbe met purely thanks to non-permanent sources of revenue and withoutany expenditure reform. For 2010-11, Mukherjee will also be helpedby a sharper-than-expected rebound in agriculture from a bad year -statisticians call it the 'base effect' - and consumption demandthat will likely buoy the rate of economic growth for 2010-11 to theprojected 8.5 per cent or close to it.

But the real worry is that the industrial and services sectorshave performed below expectations and may not look up unless thereare strong positive signals. "The threat (of a slowdown) is real.The existing momentum could lose steam quickly," says Ajit Ranade,Chief Economist, Aditya Birla Group. "Steep hikes in interest ratesnow could spook growth more than inflation as the time to beproactive is past." Foreign direct investment is down 27 per cent to$19 billion in 2010 while rivals such as China have increased theirinflows above $100 billion.

The spotlight of expectations then shifts firmly to New Delhi. Ifthe Congress-led United Progressive Alliance, or UPA, governmentdoes get its act together, the outlook could recover as quickly asit worsened. If that happens, it could make for a return to the pre-global financial crisis rate of growth as investors begin to resumefunding of capacity expansions - which is also the prescribedantidote to inflation. Prime Minister Manmohan Singh enjoys a hard-tomatch reputation with investors. Even the slightest push fromSingh to the Cabinet could improve the investment sentiment.

But Singh is showing no signs of emerging from the stupor the 2Gspectrum scandal and the Andimuthu Raja episode cast him into lastyear. Analysts point out to a telltale sign in the mid-JanuaryCabinet reshuffle: Singh did not drop the poor performers.

A V Dharmakrishnan

ED (Finance), Madras Cements

Coal prices have more than doubled in two years to $140 per tonneand power tariff has risen by 10 per cent, besides interest ratehikes.

Impact: In addition to increased costs, cement makers in SouthIndia face demand contraction and a glut in manufacturing capacity

In a world run by realpolitik, Cabinet colleagues and key allieslike Agriculture Minister Sharad Pawar are clearly untouchabledespite repeatedly not reading inflationary signals right or takingsteps beyond Band-Aid fixes like export curbs. Deeper supply chainand distribution bottlenecks have just not been addressed.

Cracks in the Cabinet under a leader perceived as not decisiveand strong came out in the open when Commerce and Industry MinisterAnand Sharma, worried about the impact of the rate hikes onindustrial growth, wrote to Mukherjee a couple of weeks ago. "Thehigh inflation in primary articles, particularly vegetables is moreon account of supplyside constraints and monetary policy may not bethe most suitable intervention to deal with the situation," heargued. Mukherjee did not reply.

Little is coming by way of signals from the government on itsstrategy on the economy. Mukherjee held two press conferences in thelast two weeks of January but said precious little. Two bigquestions loom before businessmen and the Indian workforce: how badcould matters get for the economy and growth in gross domesticproduct? And, how long could the rough patch last? Three of the fiveeconomists BT spoke with ) predict GDP growth will be less than 8.2per cent in 2011-12. Even the RBI, which has retained its 8.5 percent estimate for 2010-11 with an upward bias, is cautious on theoutlook for the next financial year saying "growth may declinesomewhat" due to a high base effect this time for agriculture.

These predictions will come true, if not next year then the yearafter for there are glaring gaps in the economy that need fixing.Agriculture and food policy, for instance. V.S. Vyas, Member of theEconomic Advisory Council to the Prime Minister, says there aredeficiencies at multiple levels: minimum support prices, and theinstitutional system for transfer of technology, benefits ofresearch, seeds and inputs. "The extension machinery is completelybroken down. Big farmers have some access but not small farmers.Even 50 per cent of the credit meant for small farmers is notreaching them," he says. "Going forward, two or three things need tobe looked at - macroagri-policy, region-specific agri-policy andwater management." Put simply, goals that cannot be achievedovernight or without political will.

R. Seshasayee

Managing Director, Ashok Leyland

In December, we had ramped up our production in Pantnagar andsoon faced serious hurdles. Roads were not taking the load. We couldnot get our supplies in nor could we move out the fully builtchassis.

Impact: Until Sept 2010, 118 roads and highway projects - 37% oftotal projects - were running behind schedule. As a result, fewerthan 4 km of roads were built a day compared to the 21-km target

In the non-food economy, increasing the pace of infrastructurerollout is a difficult target given that project completion comeswith significant time lags - three years after a road project, forinstance, is awarded to a developer. By that yardstick, the lowmeasure of road projects awarded in 2008-09 - 643 km - will come tohaunt the UPA this fiscal year and the next. The road projectsawarded in 2009-10 and to date in 2010-11 are a healthy 3,360 km and3,860 km, respectively, but they are caught in poor projectexecution, and time and cost overruns, partly due to problems overland acquisition and environmental approvals.

Ask Ajit Gulabchand, Chairman and Managing Director of HindustanConstruction Company, or HCC, who has acquired a poster-boy statusover his fights with the government over green clearance for hisunder-construction hill station at Lavasa. The Rs 3,000-croreproject is one among the 64 Environment Minister Jairam Ramesh hashalted. Prior approvals to another 469, some dating back to 2006,are at various stages of review - amounting to about one projecthalted a day since Ramesh got his job. Even the HCC chairman doesnot grudge environment standards but wants consistency in policy."We are losing Rs 2 crore a day. India should have environmentstandards in place rather than arbitrary action against projects,"he insists.

In spite of the latest hiccups, few dispute a sound future of theIndian economy in the long term. If anything, what worriesprofessionals like Akhil Gupta, Chairman and Managing Director ofbuyout fund Blackstone in India, is whether the high inflation willchange from one of cyclical nature to "a structurally highinflation". Or, whether India will chug along but will have to livewith high inflation, a la Latin American economies. The fix forthis, he recommends in an echo of the RBI, is fiscal discipline.

Whether India will get to tackle its troubles and stokeinvestment back to the levels seen a few years back is anybody'sguess. One instance from the Congress Plenary meeting at Burari, atown northwest of New Delhi, a week before Christmas does notinspire confidence. Party President Sonia Gandhi's call to hersenior leaders and party workers to clean up corruption, especiallyaround land acquisition, was greeted with silence.

"So, no clapping for this one," asked a surprised Gandhi. Thatsurprise, showing the disconnect between the Congress leadership andits rank and file, is not good news for business.

Additional reporting by Rishi Joshi, Manu Kaushik, N. Madhavan,K. R. Balasubramanyam, Anand Adhikari, Shamni Pande, Suman Layak andT.V. Mahalingam

British army dismantles parcel bomb in Northern Ireland's mail-sorting center

British army experts dismantled a parcel bomb Wednesday in Northern Ireland's major mail-sorting center, which had to be evacuated for six hours.

The bomb interception came two weeks after Northern Ireland police chief Hugh Orde put the British territory on alert against the possibility of increased attacks by Irish Republican Army dissidents. Police have resumed road checkpoints to deter the threat of car bombs.

The Police Service of Northern Ireland said in a statement that postal workers discovered the bomb at the main Royal Mail sorting center near Belfast, which uses high-tech X-ray and other scanning devices. The statement described the bomb as "viable and unstable."

Officials at the police press office in Belfast declined to disclose the delivery address on the package.

In the past, both IRA dissidents and anti-Catholic extremists have tried to target their political enemies using bombs in the mail. Most of them have been detected at the suburban Belfast sorting center.

IRA dissidents opposed to the group's 2005 decision to renounce violence and disarm have continued to mount sporadic attacks in pursuit of the traditional IRA goal of forcing Northern Ireland out of the United Kingdom.

Dissidents most recently claimed responsibility for shooting two off-duty policemen in separate attacks in November; both officers, struck by shotgun blasts as they drove their private cars, survived.

Staff transitions at MC Canada

Winnipeg, Man.

Four staff persons at MC Canada are making transitions this summer.

Kathy Giesbrecht, a long-time friendly voice on the phone at MC Canada's Resource Centre will end her half-time work there this summer. She will continue her youth ministry at Springstein (Manitoba) Mennonite Church. Giesbrecht was recently ordained by her faith community at Springstein and re-affirmed in her work there. She has been working at the Resource Centre for six years, and juggling her youth minister's position at Springstein.

"My greatest joy has been the privilege of talking with people and hearing what is happening in their congregations, this has infused me with much hope for the days ahead of us," said Giesbrecht. She will leave her post on August 1, but will be around to assist with transition to her successor(s).

Connie Loeppky will also end her half-time position at the Resource Centre. Loeppky has faithfully served many congregations and their worship leaders in various capacities at the centre since 1990.

Loeppky says that she made a commitment one year ago to re-evaluate her plans for leaving in June 2003. She adds, "It has been a privilege to be part of God's ministry by relating to many people via phone, e-mail, letter, and in person. It has been a vocation that never felt like a job but rather a service that I enjoyed being part of." She is looking forward to exploring the options that an unencumbered future brings. Her last day will be June 30.

Anne Campion, director of youth ministries for MC Canada made a commitment to re-evaluate her role when she accepted her position in February of 2002. "This has been a full and exciting year," said Campion. "My roles of half-time associate pastor, director of youth ministry for MC Canada, and mother of two busy teens has proved to be an immense juggling act." Campion fulfills her pastoral role at Avon Mennonite Church in Stratford, Ont. "I'll really miss the energy of the youth in our churches across Canada, and networking with various youth leaders, and conference youth ministers from different regions," she said. Anne completes her service with MC Canada August 31, 2003.

Isbrand Hiebert, longtime editor of the German language newspaper Der Bote is pursuing medical treatment for a chronic health problem. Ingrid Janzen Lamp of Swift Current, Saskatchewan, is currently filling the role as interim editor.

Justina Heese, executive secretary of MC Canada's Christian Formation area, said, "I am grateful for the faithful service and vibrant ministries of these individuals. They are a gift from God."--MC Canada release

Lester throws 57 pitches, says no deal is done

Jon Lester had much more to say about his changeup than he did about his future.

The left-hander threw 57 pitches in his third spring training start, and a makeshift Boston Red Sox lineup outslugged the Pittsburgh Pirates 15-14 on Monday in a 10-inning game that lasted 4 hours, 10 minutes.

Lester's outing came one day after Yahoo.com reported that he had agreed to a $30 million, five-year contract extension with a $13 million club option for 2014.

The Yahoo! Sports report cited an unidentified source close to the team. Lester, however, said no deal had been completed.

Wimbledon unveils Centre Court roof to public

Wimbledon picked the perfect day for showcasing its new retractable roof on Centre Court.

The sliding, canvassed roof barely had time to shut Sunday for the All England Club's grand opening _ or closing _ of the new structure before the rain starting pouring down over south London. In the same kind of weather that has forced so many rain delays in the past, the Centre Court then hosted a televised tennis spectacle in perfect conditions.

"It's a real treat to be able to play in these conditions inside, when it's been so miserable, cold, windy and wet outside," said Tim Henman, who teamed with Kim Clijsters to play married couple Andre Agassi and Steffi Graf in doubles and singles matches.

It seems no fan with a Centre Court ticket will ever return home again without seeing a single shot of tennis played.

"Fundamentally, it worked like it was supposed to work," said Ian Ritchie, the All England Club's chief executive.

To kick off the event, the sliding roof slowly started unfolding with a soft whirring in an afternoon ceremony as British singers Faryl Smith and Katherine Jenkins sang "Amazing Grace" in the center of the court.

The 15,000 spectators _ along with invited guests such as former tennis great Boris Becker _ collectively raised their heads and cameras as the 10 trusses supporting a translucent canvas slowly covered the court to shield it from the heavy gray clouds above.

As the two-parted roof closed without a hitch, it was greeted by a thunderous applause.

Then shortly after the tennis began, as if on cue, it started to rain.

Not that anyone inside the stadium noticed.

"The conditions were really good," Clijsters said. "And I love the sound. Wimbledon already had that, where you feel like when (the crowd is) really into the match, the sound really comes down to the players. And now even more so with the roof. For the players, it just feels like they're right there next to you."

Agassi also said the closed surrounding will take the atmosphere to a new level.

"The sound was magnificent," he said. "I think when you get two people out there who can really play, and move and hit the ball, I think you're going to feel a level of titanic battle that you haven't seen yet. ... That's an environment that lends itself to some spectacular tennis."

The roof is perhaps the biggest change to come to the 87-year-old tennis cathedral, and marks a remarkable break with tradition by the famously conventional All England Club.

But if the Centre Court looked more modern than ever, the action on the grass looked decidedly old-school.

Graf, a seven-time Wimbledon champion, returned to the Centre Court for the first time in 10 years, while Agassi won his first Grand Slam title here in 1992.

They still know how to entertain a crowd, though. The couple drew a large cheer by exchanging a good-luck kiss on the court before the doubles match. Then they showed they can still make some shots as well.

On his first serve, Agassi taunted Clijsters for standing close to the baseline for her return, telling her to "move back a bit." When she didn't, he punished her with an ace.

On the next serve, Henman moved nearly all the way back to the wall for his return.

The ladies had their moments as well. In the third game, Clijsters and Graf exchanged so many crosscourt forehands that Henman and Agassi just put their hands on their hips and watched. When Clijsters finally hit the ball Agassi's way, he netted a volley _ to Graf's feigned dismay.

In the end, Henman and Clijsters won the one-set match 7-6 (6) after Agassi hit a lob long. Agassi then beat Henman 6-4 in singles and Clijsters defeated Graf by the same score.

But as much as the crowd enjoyed seeing the old Wimbledon stalwarts back on the Centre Court, it was the new structure that received the most praise.

"It's beautiful," said third-ranked Andy Murray, who hopes to become the first Briton since Fred Perry in 1936 to win Wimbledon. "It looks very nice, compared to most roofs."

Five-time Wimbledon champion Roger Federer, who beat rival Rafael Nadal in the final of the Madrid Open on Sunday, sent a recorded greeting to the crowd, saying he couldn't wait to play at the new-look Centre Court.

"So often do we just wait around (during rain delays)," Federer said. "I know that can be fun too, but maybe after a few days you want to see a match. ... For me it's nice to see that Wimbledon is taking such a big step in the right direction."

The roof takes about 10 minutes to close, and the Centre Court's ventilation system then needs about 30 minutes to get moisture out of the air and create the right conditions. During short rain showers, organizers still plan on using the traditional covers on the court to create shorter breaks. Once the roof is up, it will not open up until a match is over.

After finally getting the roof completed, Ian Ritchie, the chief executive of the All England Club, said he wouldn't be surprised to see perfect weather at this year's tournament. But that's just fine with him.

"We see this as an outdoor summer tournament," he said. "The roof is an insurance."