Autochthonous buffalo-gut origin Pediococcus pentosaceus RM119 decreased diarrhea and enhanced gut health, immunity in neonatal Murrah calves.

This study assessed the effect of autochthonous probiotic Pediococcus pentosaceus RM119 on gut health, growth, and nutrient utilization in calves. Twelve buffalo calves (<15 d) were divided into two groups, control without probiotics, and probiotic group with P. pentosaceus RM119 @ 108 CFU/calf/d. The probiotic group showed a reduction (p < 0.05) in fecal score, diarrhea episodes and duration of diarrhea. The fecal pH, fecal ammonia was lower, whereas lactate was higher in probiotic group than control. There was a significant increase (p < 0.01) in the concentration of fecal acetate, propionate and butyrate levels in the probiotic supplemented group. The fecal lactobacilli and bifidobacterium were higher (p < 0.01), whereas, fecal coliform and clostridial count were lower (p < 0.01) in P. pentosaceus RM119 supplemented group. There was an improvement in reduced glutathione anti oxidant. Overall, buffalo-gut origin P. pentosaceus RM119 reduced the frequency and severity of diarrhea in neonatal buffalo calves and improved the gut health.

Microencapsulated and Lyophilized Lactobacillus acidophilus Improved Gut Health and Immune Status of Preruminant Calves.

The present study was conducted to study the effect of microencapsulated, lyophilized, or fermented milk using Lactobacillus acidophilus NCDC15 as a probiotic to improve gut health, growth, nutrient utilization, and immunity status of young crossbred calves. The viable culture of L. acidophilus was used for preparation of different probiotic forms/products. To compare the efficacy of probiotic products, twenty crossbred calves (3-day old) were divided into four groups (n = 5), control (C), fed only milk and basal diet, and treatment groups, supplemented with microencapsulated, fermented, and lyophilized probiotic at 108 colony-forming units, respectively. Probiotic-supplemented groups showed reduction in faecal score, faecal pH, and ammonia concentration as compared to control indicating decreased diarrheal incidence. There was an increase (P < 0.05) in the concentration of faecal lactate and butyrate in the probiotic-supplemented groups. The faecal count (log10 (CFU)/g of fresh faeces) of lactobacilli and bifidobacteria was higher (P < 0.05), whereas faecal coliforms and clostridia count were reduced (P < 0.001) in all the probiotic fed groups as compared to control. The cell-mediated immunity was improved (P < 0.05) in the microencapsulated and fermented probiotic groups. However, there was no effect on the nutrient utilization, average daily gain, and blood biochemical profile. Therefore, it is concluded that the fermented, microencapsulated and lyophilized probiotic products were superior in improving the gut health in terms of its microbiota and metabolites and cell-mediated immunity response in calves, irrespective of form of probiotic. The increased population of Lactobacillus and Bifidobacterium decreased the colonization of the gut by pathogens such as Escherichia coli and Clostridium by exclusion and production of organic acids in the intestine. This decreased the diarrhoeal incidence (1.3 vs 1.8) and days in diarrhoea (3.9 vs 5.8) in calves in probiotic fed groups as compared to control.

Insights into Metatranscriptome, and CAZymes of Buffalo Rumen Supplemented with Blend of Essential Oils.

The aim of this study was to investigate the rumen microbial diversity and functionality in buffaloes fed with a blend of essential oils (BEO) using LSD switch over design. The BEO consisting of blend of Trachyspermum copticum (Ajwain) oil, Cymbopogon citratus (lemon grass) oil and Syzygium aromaticum (clove bud) oleoresin mixed in equal proportion, was fed at the rate of 0, 0.75 and 1.5 ml/100 kg of body weight in 0 (control), 0.75 and 1.5 groups, respectively. The metatranscriptomic libraries of the rumen microbiome were represented by 7 domains, 84 phyla, 64 archeal genera and 663 bacterial genera with Bacteroidetes and Firmicutes constituting 80% of phyla abundance irrespective of feeding regime. Methanogenic archaea was represented by 22 phyla with Methanobrevibacter as the major genus. BEO feeding reduced the abundance of Methanococcus and Thermoplasma (P < 0.05) at all levels. The results revealed that the feeding of BEO shifted the archeal and bacterial population at very low magnitude. The study explored the vast diversity of buffalo rumen bacteria and archaea, and the diverse wealth of rumen enzymes (CAZymes), which revealed that a major part of CAZymes comes from the less known rumen microbes indicating alternative paths of fiber degradation along with the very well known ones.

Understanding the relationship between soil properties and litter chemistry in three forest communities in tropical forest ecosystem.

In this study, we investigated the relationship between soil properties and litter chemistry in three forest communities, i.e., Sal mixed forest (SMF), dry mixed forest (DMF), and teak plantation forest (TPF), in tropical deciduous forest ecosystem in North India. Fresh leaf litter and soil samples were collected at two soil depths (0-15 and 15-30 cm) from all these three forest communities. Litter bag experiment was also conducted to know differences in litter nutrients after its decomposition. The concentrations (mg kg-1) of different nutrients such as sodium (Na) 2.6, potassium (K) 38.5, calcium (Ca) 425, and carbon (C) 45.54% were highest in fresh litter collected from DMF. Total organic carbon (g kg-1) was significantly higher in SMF (19.23) in comparison to DMF (18.41) and TPF (13.61) at 0-15-cm soil depth. Na, K, Ca, available P, total P, available N, and total N were highest in DMF soil. We observed significantly positive correlation between all nutrients of litter and soil. Although soil bulk density (BD) and particle density (PD) showed their significant negative correlation with litter C, total porosity was positively correlated. Similarly, litter Na has its significant negative correlation with BD and positive correlation with PD. The litter chemistry played a significant role in changing soil pH and TOC. All litter nutrients, except total P, have their significant positive correlation with soil pH. Total P, C, and N of litter have their significant positive correlation with total soil organic carbon. This indicates that litter chemistry and soil properties have specific relation among them despite unique species composition in each forest community.

Interaction of abiotic factor on soil CO2 efflux in three forest communities in tropical deciduous forest from India.

Environmental factors along with soil physico-chemical properties play a significant role on the diurnal trend of soil CO2 efflux. Soil CO2 efflux in Indian tropical forests is poorly studied. We studied the soil CO2 efflux in a representative tropical deciduous forest at Katerniaghat Wildlife Sanctuary (KWLS), Uttar Pradesh. The three forest communities namely dry mixed (DMF), Sal mixed (SMF), and Teak plantation (TPF) were selected for measuring soil CO2 efflux in the summer season during April to May 2017 using automated LI-COR 8100 soil CO2 flux system. Soil physico-chemical parameters were also studied in the three abovementioned forest communities. We also measured the different microclimatic variables at forest understorey in all three communities during the summer season. Total day time soil CO2 efflux of 826.70, 1089.24, and 828.94 (µmolCO2 m-2d-1) was observed in TPF, SMF, and DMF respectively. Soil CO2 efflux observed significant differences (P < 0.01) among the three forest communities studied for the summer season in tropical deciduous forest of Terai Himalaya. Average soil CO2 efflux rate (µmol CO2 m-2 s-1) of 4.06 ± 0.36, 5.03 ± 0.45, and 4.37 ± 0.79 was observed in TPF, SMF, and DMF, respectively, which is positively correlated with total organic carbon (TOC) and water holding capacity (WHC) among soil physico-chemical variables. Among microclimatic variables, soil temperature (ST, °C) and air temperature (AT, °C) observed strong positive correlation with day time soil CO2 efflux in all three communities. Significant increase in soil CO2 flux was observed with increasing air and soil temperature (AT and ST) in DMF and SMF. Maximum TOC of 19.23 g Kg-1 was observed in SMF among all communities in the summer season. The result showed that soil CO2 efflux is closely associated with TOC, WHC, AT, and ST for Indian deciduous forest ecosystems.

Impact of collected sunlight on ZnFe2O4 nanoparticles for photocatalytic application.

In the present investigation, a series of zinc ferrite (ZnFe2O4) nanoparticles were synthesized using a facile, reproducible and scalable chemical co-precipitation route for sunlight assisted photocatalytic degradation application. In the present work, we have prepared ZnFe2O4 with 1:1, 1:2 and 1:3 M ratio of zinc chloride and ferric chloride respectively. This work reports the photodegradation of organic methylene blue dye molecules using ZnFe2O4 under both normal sunlight, and collected sunlight. Among other annealing temperatures, particularly the ZnFe2O4 annealed at 600 °C with a molar ratio of 1:3 showed the highest photocatalytic degradation of methylene blue. Interestingly close to 99% degradation in less than 60 min of collected sunlight illumination has been achieved indicating maximum photocatalytic activity under investigation. This expounding study will open new way of light harvesting in the field of photocatalysis which is different from common praxis.

Effect of feeding of blend of essential oils on methane production, growth, and nutrient utilization in growing buffaloes.

OBJECTIVE: An experiment was conducted to study the effect of a blend of essential oils (BEO) on enteric methane emission and growth performance of buffaloes (Bubalus bubalis). METHODS: Twenty one growing male buffaloes (average body weight of 279±9.3 kg) were divided in to three groups. The animals of all the three groups were fed on a ration consisting of wheat straw and concentrate mixture targeting 500 g daily live weight gain. The three dietary groups were; Group 1, control without additive; Group 2 and 3, supplemented with BEO at 0.15 and 0.30 mL/kg of dry matter intake (DMI), respectively. RESULTS: During six months feeding trial, the intake and digestibility of dry matter and nutrients (organic matter, crude protein, ether extract, neutral detergent fibre, and acid detergent fibre) were similar in all the groups. The average body weight gain was tended to improve (p = 0.084) in Group 2 and Group 3 as compared to control animals. Feeding of BEO did not affect feed conversion efficiency of the animals. The calves of all the three groups were in positive nitrogen balance with no difference in nitrogen metabolism. During respiration chamber studies the methane production (L/kg DMI and L/kg digestible dry matter intake was significantly (p<0.001) lower in Group 2 and Group 3 as compared to control animals. CONCLUSION: The results indicated that the BEO tested in the present study have shown potential to reduce enteric methane production without compromising the nutrient utilization and animal performance and could be further explored for its use as feed additive to mitigate enteric methane production in livestock.

Effect of high and low roughage total mixed ration diets on rumen metabolites and enzymatic profiles in crossbred cattle and buffaloes.

AIM: A comparative study was conducted on crossbred cattle and buffaloes to investigate the effect of feeding high and low roughage total mixed ration (TMR) diets on rumen metabolites and enzymatic profiles. MATERIALS AND METHODS: Three rumen-fistulated crossbred cattle and buffalo were randomly assigned as per 3×3 switch over design for 21-days. Three TMR diets consisting of concentrate mixture, wheat straw and green maize fodder in the ratios of (T1) 60:20:20, (T2) 40:30:30, and (T3) 20:40:40, respectively, were fed to the animals ad libitum. Rumen liquor samples were collected at 0, 2, 4, 6, and 8 h post feeding for the estimation of rumen biochemical parameters on 2 consecutive days in each trial. RESULTS: The lactic acid concentration and pH value were comparable in both species and treatments. Feed intake (99.77±2.51 g/kg body weight), ruminal ammonia nitrogen, and total nitrogen were significantly (p<0.05) higher in buffalo and in treatment group fed with high concentrate diet. Production of total volatile fatty acids (VFAs) was non-significant (p>0.05) among treatments and significantly (p<0.05) greater in crossbred cattle than buffaloes. Molar proportions of individual VFAs propionate (C3), propionate:butyrate (C3:C4), and (acetate+butyrate):propionate ([C2+C4]:C3) ratio in both crossbred cattle and buffalo were not affected by high or low roughage diet, but percentage of acetate and butyrate varied significantly (p<0.05) among treatment groups. Activities of microbial enzymes were comparable among species and different treatment groups. A total number of rumen protozoa were significantly (p<0.05) higher in crossbred cattle than buffaloes along with significantly (p<0.05) higher population in animal fed with high concentrate diet (T1). CONCLUSION: Rumen microbial population and fermentation depend on constituents of the treatment diet. However, microbial enzyme activity remains similar among species and different treatments. High concentrate diet increases number of rumen protozoa, and the number is higher in crossbred cattle than buffaloes.

Impact of levels of total digestible nutrients on microbiome, enzyme profile and degradation of feeds in buffalo rumen.

The present study was aimed at understanding a shift in rumen microbiome of buffaloes fed various levels of total digestible nutrients. To understand the process, the metagenomics of rumen microbes, in vivo and in vitro rumen fermentation studies were carried out. Three rumen fistulated adult male Murrah buffaloes were fed three isonitrogenous diets varying in total digestible nutrients (70, 85 and 100% of TDN requirement) in 3X3 switch over design. On dry matter basis, wheat straw/ roughage content were 81, 63 and 51% and that of maize grain was 8, 16 and 21% in three diets respectively. After 20 d of feeding, rumen liquor and rumen contents were sampled just before (0h) and 4h post feeding. Ruminococcus flavefaciens and R. albus (estimated with real time PCR) were higher in high roughage diets. The predominant phyla in all the three groups were Bacteroidetes, Firmicutes followed by Proteobacteria, Actinobacteria and Fibrobacteres. A core group of more than fifty rumen bacteria was present in all the animals with very little variations due to level of TDN. The most predominant bacterial genera reported in order of decreasing abundance were: Prevotella, Bacteroides, Clostridium, Ruminococcus, Eubacterium, Parabacteroides, Fibrobacter, Butyrivibrio etc. The higher diversity of the enyzmes families GH 23, GH 28, GH 39, GH 97, GH 106, and GH 127 (the enzymes active in fibre and starch degradation) were significantly higher on 100%TDN diet while CE 14 (required for the hydrolysis of bond between carbohydrate and lignin) was higher on low TDN (70%) diet, indicating ester bond cleavage was better in animals fed high roughage (wheat straw) diet.

Effect of feeding tannin-degrading bacteria Streptococcus gallolyticus strain TDGB 406 on meat quality of goats fed with Quercus semicarpifolia leaves.

The effect of feeding tannin-degrading bacteria (Streptococcus gallolyticus strain TDGB 406) on carcass characteristics of goats fed with oak (Quercus semicarpifolia) leaves was studied on 18 male goats (4 months old, average body weight 9.50 ± 1.50 kg), distributed into three groups of six animals each. The animals of group 1 served as control, while the animals of groups 2 and 3 were given (at 5 ml/kg live weight) autoclaved and live culture of isolate TDGB 406 (10(6) cells/ml), respectively. The animals were fed with oak leaves as a basal roughage source and maize hay along with fixed quantity of concentrate mixture. After 4 months of feeding, the animals were slaughtered for carcass studies. The feeding of live culture of isolate TDGB 406 did not cause any effect (P > 0.05) on pre-slaughter weight, empty body weight, carcass weight, dressing percent, and yield of wholesale cuts (neck, rack, shoulder, breast, shank, loin, leg, and flank) of the goat meat. The chemical composition of longissimus dorsi muscle was comparable (P > 0.05) among the groups. The organoleptic evaluation of pressure-cooked meat in terms of tenderness and overall palatability was increased significantly (P < 0.05) in the meat of group 3 where live culture was supplemented. The other attributes were similar among the groups. It was concluded that supplementation of tannin-degrading bacteria S. gallolyticus strain TDGB 406 to goats fed with oak leaves did not affect the carcass characteristics and meat quality.

Effect of feeding tannin degrading bacterial culture (Streptococcus gallolyticus strain TDGB 406) on nutrient utilization, urinary purine derivatives and growth performance of goats fed on Quercus semicarpifolia leaves.

To study the effect of supplementation of tannin degrading bacterial culture (Streptococcus gallolyticus strain TDGB 406) on growth performance, nutrient utilization and urinary purine derivatives of goats fed on oak (Quercus semicarpifolia) leaves. For growth study, eighteen billy goats (4 month old, average body weight 9.50 ± 1.50 kg) were distributed into three groups of six animals each. The animals of group 1 served as control while animals of groups 2 (T1) and 3 (T2) were given (@ 5 ml/kg live weight) autoclaved and live culture of isolate TDGB 406 (10(6) cells/ml) respectively. The animals were fed measured quantity of dry oak leaves as the main roughage source and ad libitum maize hay along with fixed quantity of concentrate mixture. The feeding of live culture of isolate TDGB 406 (probiotic) did not affect dry matter intake and digestibility of nutrients except that of dry matter and crude protein, which was higher in T2 group as compared to control. All the animals were in positive nitrogen balance. There was no significant effect of feeding isolate TDGB 406 on urinary purine derivatives (microbial protein production) in goats. The body weight gain and average live weight gain was significantly higher (p = 0.071) in T2 group as compared to control. Feed conversion efficiency was also better in the goats fed on live culture of TDGB 406 (T2). The feeding of tannin degrading bacterial isolate TDGB 406 as probiotic resulted in improved growth performance and feed conversion ratio in goats fed on oak leaves as one of the main roughage source.

Is hematopoietic cell transplantation still a valid option for mantle cell lymphoma in first remission in the chemoimmunotherapy-era?

Modern chemoimmunotherapies have produced higher response rates and improved survival in mantle cell lymphoma (MCL); however, disease relapse remains a challenge. The availability of various post-remission maintenance or consolidation strategies, have led some to question the role of upfront autologous hematopoietic cell transplantation (auto-HCT) consolidation for MCL, in the chemoimmunotherapy-era. A one size fits all approach is no longer appropriate for MCL in first remission, and the choice of preferred post-remission (observation, maintenance or consolidation) strategy is increasingly becoming a factor of patient age, comorbidities and disease risk stratification. In select low-risk patients (based on Mantle cell lymphoma International Prognostic Index (MIPI)), observation following rituximab plus Hyper-CVAD-like inductions seems appropriate. Rituximab maintenance after anthracycline-based chemoimmunotherapies in elderly transplant ineligible patients has shown survival benefit and should be considered a valid option. Limited studies suggest feasibility of radioimmunotherapy consolidation in first remission; however, in the absence of randomized data, this modality remains investigational. In younger, transplant-eligible patients receiving cytarabine-containing inductions, upfront consolidation with auto-HCT has shown survival benefit, and remains a standard-of-care option in the modern-era. Hyper-CVAD associated stem cell mobilization failure is an increasingly recognized problem, underscoring the need for alternative inductions, or consideration for early stem cell collection, when this induction regimen is used. Outcomes of high-risk MIPI patients remain suboptimal with currently available induction and post-remission strategies and represents an area where adoptive immunotherapy in the form of allogeneic-HCT warrants investigation. Incorporation of novel MoAbs and targeted agents (PI3K inhibitors, mTOR inhibitors, BTK inhibitors and so on.) in maintenance and consolidation strategies will build on the significant therapeutic gains of last decade, in coming years.

Differential growth factor control of bone formation through osteoprogenitor differentiation.

The osteogenic factors bone morphogenetic protein (BMP-7), platelet-derived growth factor (PDGF)-BB, and fibroblast growth factor (FGF-2) regulate the recruitment of osteoprogenitor cells and their proliferation and differentiation into mature osteoblasts. However, their mechanisms of action on osteoprogenitor cell growth, differentiation, and bone mineralization remain unclear. Here, we tested the hypothesis that these osteogenic agents were capable of regulating osteoblast differentiation and bone formation in vitro. Normal human bone marrow stromal (HBMS) cells were treated with BMP-7 (40 ng ml(-1)), PDGF-BB (20 ng ml(-1)), FGF-2 (20 ng ml(-1)), or FGF-2 plus BMP-7 for 28 days in a serum-containing medium with 10 mM beta-glycerophosphate and 50 microg ml(-1) ascorbic acid. BMP-7 stimulated a morphological change to cuboidal-shaped cells, increased alkaline phosphatase (ALKP) activity, bone sialoprotein (BSP) gene expression, and alizarin red S positive nodule formation. Hydroxyapatite (HA) crystal deposition in the nodules was demonstrated by Fourier transform infrared (FTIR) spectroscopy only in BMP-7- and dexamethasone (DEX)-treated cells. DEX-treated cells appeared elongated and fibroblast-like compared to BMP-7-treated cells. FGF-2 did not stimulate ALKP, and cell morphology was dystrophic. PDGF-BB had little or no effect on ALKP activity and biomineralization. Alizarin Red S staining of cells and calcium assay indicated that BMP-7, DEX, and FGF-2 enhanced calcium mineral deposition, but FTIR spectroscopic analysis demonstrated no formation of HA similar to human bone in control, PDGF-BB-, and FGF-2-treated samples. Thus, FGF-2 stimulated amorphous octacalcium phosphate mineral deposition that failed to mature into HA. Interestingly, FGF-2 abrogated BMP-7-induced ALKP activity and HA formation. Results demonstrate that BMP-7 was competent as a sole factor in the differentiation of human bone marrow stromal cells to bone-forming osteoblasts confirmed by FTIR examination of mineralized matrix. Other growth factors, PDGF, and FGF-2 were incompetent as sole factors, and FGF-2 inhibited BMP-7-stimulated osteoblast differentiation.

Eubacterium pyruvativorans sp. nov., a novel non-saccharolytic anaerobe from the rumen that ferments pyruvate and amino acids, forms caproate and utilizes acetate and propionate.

Two similar gram-positive rods were isolated from 10(-6) dilutions of ruminal fluid from a sheep receiving a mixed grass hay/concentrate diet, using a medium containing pancreatic casein hydrolysate as sole source of carbon and energy. The isolates did not ferment sugars, but grew on pyruvate or trypticase, forming caproate as the main fermentation product and valerate to a lesser extent. Acetate and propionate were utilized. One of these strains, I-6T, was selected for further study. Strain I-6T was a non-motile coccal rod, 1.2 x 0.4 microm, with a gram-positive cell wall ultrastructure and a G + C content of 56.8 mol%. No spores were visible, and strain I-6T did not survive heating at 80 degrees C for 10 min. Its rate of NH3 production was 375 nmol (mg protein)(-1) min(-1), placing it in the 'ammonia-hyperproducing' (or HAP) group of ruminal bacteria. 16S rDNA sequence analysis (1296 bases) indicated that it represents a novel species within the 'low-G + C' gram-positive group, for which the name Eubacterium pyruvativorans sp. nov. is proposed. Among cultivated bacteria, strain I-6T was most closely related (89% identity) to other asaccharolytic Eubacterium isolates from the mouth and the rumen. It was 98% identical to uncultured bacterial sequences amplified by others from ruminal digesta.

Microbial status and rumen enzyme profile of crossbred calves fed on different microbial feed additives.

AIMS: To test various microbial cultures as cattle feed additives. METHODS AND RESULTS: Four groups of newly born crossbred calves (average body weight 23.5 kg) were reared on green berseem and calf starter which was devoid of cereal grains. Milk was fed up to 8 weeks of age, starting with one tenths and gradually reducing to one twentieths of the body weight. One hundred millilitres of microbial feed additive or 100 g fermented feed was fed to the animals of group 2 (curd containing lactic acid bacteria at 10(8) cfu x ml(-1)), group 3 (Saccharomyces cerevisiae NCDC-49 at 10(6) cfu x ml(-1)) and group 4 (Lactobacillus acidophilus-15 at 10(8) cfu x ml(-1)). Group 1 served as control. The incidence and duration of diarrhoea was lower in the animals of probiotic fed groups as compared to control group. Out of three microbial feed additives, yeast feeding showed maximum suppression of diarrhoea followed by Lactobacillus and curd. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: There was no effect of probiotic feeding on the log number of cells of lactic acid bacteria, yeast and coliform bacteria in the faeces and rumen liquor at any age. The activities of carboxymethylcellulase, xylanase, beta-glucosidase, alpha-glucosidase, alpha-amylase, protease, urease and pH of the rumen liquor remained unaffected by probiotic feeding at all ages tested in this experiment.

Erk is essential for growth, differentiation, integrin expression, and cell function in human osteoblastic cells.

Extracellular signal-regulated kinases (Erks), members of the mitogen-activated protein kinase superfamily, play an important role in cell proliferation and differentiation. In this study we employed a dominant negative approach to determine the role of Erks in the regulation of human osteoblastic cell function. Human osteoblastic cells were transduced with a pseudotyped retrovirus encoding either a mutated Erk1 protein with a dominant negative action against both Erk1 and Erk2 (Erk1DN cells) or the LacZ protein (LacZ cells) as a control. Both basal and growth factor-stimulated MAPK activity and cell proliferation were inhibited in Erk1DN cells. Expression of Erk1DN protein suppressed both osteoblast differentiation and matrix mineralization by decreasing alkaline phosphatase activity and the deposition of bone matrix proteins. Cell adhesion to collagen, osteopontin, and vitronectin was decreased in Erk1DN cells as compared with LacZ cells. Cell spreading and migration on these matrices were also inhibited. In Erk1DN cells, expression of alphabeta(1), alpha(v)beta(3), and alpha(v)beta(5) integrins on the surface was decreased. Metabolic labeling indicated that the synthesis of these integrins was inhibited in Erk1DN cells. These data suggest that Erks are not only essential for the growth and differentiation of osteoblasts but also are important for osteoblast adhesion, spreading, migration, and integrin expression.

The cell survival signal Akt is differentially activated by PDGF-BB, EGF, and FGF-2 in osteoblastic cells.

Stimulation of osteoblast survival signals may be an important mechanism of regulating bone anabolism. Protein kinase B (PKB/Akt), a serine-threonine protein kinase, is a critical regulator of normal cell growth, cell cycle progression, and cell survival. In this study we have investigated the signaling pathways activated by growth factors PDGF-BB, EGF, and FGF-2 and determined whether PDGF-BB, EGF, and FGF-2 activated Akt in human or mouse osteoblastic cells. The results demonstrated that both ERK1 and ERK2 were activated by FGF-2 and PDGF-BB. Activation of ERK1 and ERK2 by PDGF-BB and FGF-2 was inhibited by PD 098059 (100 microM), a specific inhibitor of MEK. Wortmannin (500 nM), a specific inhibitor of phosphatidylinositol 3-kinase ( PI 3-K), inhibited the activation of ERK1 and ERK2 by PDGF-BB but not by FGF-2 suggesting that PI 3-K mediated the activation of ERK MAPK pathway by PDGF-BB but not by FGF-2. Rapamycin, an inhibitor of p70 S6 protein kinase and a downstream target of ERK1/2 and PI 3-K, did not affect the activation of ERK1 and ERK2 by the growth factors. Furthermore, our results demonstrated that Akt, a downstream target of PI 3-K, was activated by PDGF-BB but not by FGF-2. Akt activation by PDGF-BB was inhibited by PI 3-kinase inhibitor LY294002. Rapamycin had no effect on Akt activation. Epidermal growth factor (EGF) also activated Akt in osteoblastic cells which was inhibited by LY294002 but not by rapamycin. Taken together, our data for the first time revealed that the activation of ERK1/2 by PDGF-BB is mediated by PI 3-K, and secondly, Akt is activated by PDGF-BB and EGF but not by FGF-2 in human and mouse osteoblastic cells. These results are of critical importance in understanding the role of these growth factors in apoptosis and cell survival. PDGF-BB and EGF but not FGF-2 may stimulate osteoblast cell survival.

Selection of Saccharomyces cerevisiae strains for use as a microbial feed additive.

Saccharomyces cerevisiae ITCCF 2094, NCIM 3052, 1031, 1032, NCDC 42, 45, 47, 49 and 50 were screened for their tolerance to pH 2.0-7.0, various concentrations (0.00, 0.10, 0.25 0.50 and 1.0%) of a mixture of acetic, propionic and butyric acids (70:20:10), and bile salts (0.00, 0.30, 0.60 and 0.90%). Low pH (2.0-4.0) and addition of organic acids or bile salts in the medium inhibited the growth of all the strains tested, but the percentage of inhibition was variable in the different strains of yeast. Two of the strains showing maximum tolerance, 42 and 49, were further tested for in vitro dry matter degradability (IVDMD) using green berseem, wheat straw and oat hay as substrates. Saccharomyces cerevisiae 49 enhanced the IVDMD of berseem and wheat straw whereas S. cerevisiae 42 was ineffective. Based on the results of the present experiment, S. cerevisiae NCDC 49 can be considered as the best strain which might tolerate the adverse conditions in the gastrointestinal tract when used as a live microbial feed supplement in the diet of the animals.

Extracellular-signal regulated kinase signaling pathway mediates downregulation of type I procollagen gene expression by FGF-2, PDGF-BB, and okadaic acid in osteoblastic cells.

Although basic fibroblast growth factor (FGF-2) had been shown to inhibit type I collagen gene expression in osteoblast, its inhibitory mechanism is unknown. In the present study, we investigated the underlying mechanisms by which growth factors downregulate type I collagen gene expression. Treatment of mouse osteoblastic MC3T3-E1 cells with okadaic acid (40 ng/ml), an inhibitor of phosphoserine/threonine-specific protein phosphatase and activator of ERK1/2, for 24 h and 48 h completely inhibited steady-state mRNA levels of type I collagen. FGF-2 (30 ng/ml), platelet-derived growth factor-BB (PDGF-BB), 30 ng/ml, and serum, which activate ERK mitogen-activated protein kinase (MAPK) pathway also inhibited collagen type I gene expression, suggesting that the activation of ERK pathway mediates inhibition of type I collagen mRNA. This observation was further confirmed by experiments using inhibitors of the ERK pathway (i.e., PD and U0126), which increased type I collagen mRNA in MC3T3-E1 cells, indicating that the inhibition of ERK pathway upregulates type I collagen gene expression. Low serum (0.3%) markedly increased type I collagen mRNA. MEK inhibitor PD inhibited c-fos induction by FGF-2 and PDGF-BB, suggesting that c-fos is the downstream target of ERK pathway. Our data have clearly demonstrated for the first time that the ERK MAPK pathway play an important role in the regulation of type I collagen gene expression in osteoblastic cells. Results also showed that one of the mechanisms by which FGF-2 and PDGF-BB downregulate type I collagen gene expression in the osteoblast is through the activation of ERK signaling pathway.

Appendicectomy under local anaesthesia.

OBJECTIVE: To analyse our results of appendicetomy under local anaesthesia. DESIGN: Prospective study. SETTING: University hospital, India. SUBJECTS: 165 patients who presented with appendicitis between January 1996 and December 1997. INTERVENTION AND MAIN OUTCOME MEASURES: Appendicectomy after infiltration of local anaesthetic. No patient required general or spinal anaesthesia. RESULT: Five patients (3%) developed postoperative wound infections. Mean operating time was 20 minutes (range 15-30) and median hospital stay 2 days (range 1-3). CONCLUSION: Appendicectomy under local anaesthesia is quick, cost-effective and carries little morbidity. It can be safely used for all age groups.