Zinc solubilization and organic acid production by the entomopathogenic fungus, Metarhizium pingshaense sheds light on its key ecological role in the environment.

We report for the first-time higher zinc (Zn) solubilization efficiency and plant growth promotion by an entomopathogenic fungus (EPF), Metarhizium pingshaense IISR-EPF-14, which was earlier isolated from Conogethes punctiferalis, a pest of global importance. The Zn solubilizing efficiency of the fungus varied depending on the type of insoluble source of Zn used, which was observed to be 1.6 times higher in Zn3(PO4)2-amended media compared to ZnO media. In liquid media, there was a 6.2-fold increase in available Zn in ZnO-amended media, whereas a 20.2-fold increase in available Zn was recorded in Zn3(PO4)2 medium. We ascribe the production of various organic acids such as gluconic, keto-gluconic, oxalic, tartaric, malonic, succinic and formic acids, which in general, interact with insoluble Zn sources and make them soluble by forming metal cations and displacing anions as the major mechanism for Zn solubilization by M. pingshaense. However, the type and amount of organic acid produced in the media varied depending on the source of Zn used and the incubation period. Application of the fungus alone and in combination with insoluble Zn sources enhanced various plant growth parameters in rice and cardamom plants. Moreover, the uptake of Zn in rice plants was enhanced up to ~2.5-fold by fungal application. The fungus also exhibited various other plant growth-promoting traits, such as production of Indole-3-acetic acid, ammonia, siderophores, solubilization of mineral phosphate, and production of hydrolytic enzymes such as α-amylase, protease, and pectinase. Hence, apart from its use as a biological control agent, M. pingshaense has the potential to be used as a bio-fortifier to enhance the solubilization and uptake of Zn from nutrient poor soils under field conditions. Our findings shed light on the broader ecological role played by this fungus and widen its scope for utilization in sustainable agriculture.

Extracellular matrix compression temporally regulates microvascular angiogenesis.

Mechanical cues influence tissue regeneration, and although vasculature is known to be mechanically sensitive, little is known about the effects of bulk extracellular matrix deformation on the nascent vessel networks found in healing tissues. Previously, we found that dynamic matrix compression in vivo potently regulated revascularization during bone tissue regeneration; however, whether matrix deformations directly regulate angiogenesis remained unknown. Here, we demonstrated that load initiation time, magnitude, and mode all regulate microvascular growth, as well as upstream angiogenic and mechanotransduction signaling pathways. Immediate load initiation inhibited angiogenesis and expression of early sprout tip cell selection genes, while delayed loading enhanced microvascular network formation and upstream signaling pathways. This research provides foundational understanding of how extracellular matrix mechanics regulate angiogenesis and has critical implications for clinical translation of new regenerative medicine therapies and physical rehabilitation strategies designed to enhance revascularization during tissue regeneration.

Regulatory B Cells Are Reduced and Correlate With Disease Activity in Primary Membranous Nephropathy.

INTRODUCTION: Primary membranous nephropathy (PMN) is an autoimmune disease. Both T-regulatory cells (TREGs) and B-regulatory cells (BREGs) are decreased in patients with autoimmune disease. We evaluated the TREG and BREG population in patients of PMN treated with cyclical cyclophosphamide and steroid therapy (cCYC/GC). METHODS: Twenty-four patients with PMN resistant to a restrictive strategy and treated with cCYC/GC therapy and 10 healthy controls were enrolled. The proteinuria, serum creatinine, and serum albumin were tested at monthly intervals and blood samples were collected before starting cCYC/GC and at 6 and 8 (2 months wash out) months of therapy. The peripheral blood mononuclear cells (PBMCs) after staining with fluorochrome-conjugated antibodies were then subjected to flow cytometric analysis for detection of TREGs (CD3+CD4+CD25hiCD127loFoxP3+) and BREGs (CD19+CD5+CD1dhiIL10+). TREGs and BREGs are presented as the percentage of T and B cells, respectively. Cases with remission at month 18 were classified as responders, and those without any remission as nonresponders. RESULTS: Patients with PMN had a lower percentage of TREGs (P = 0.07) and BREGs compared with healthy controls (P = 0.0007). As compared with baseline, there was a significant increase in both BREGs (P = 0.001) and TREGs (P = 0.02) with the treatment (8 months). Patients who responded to therapy by 18 months had an increase in TREG (P = 0.05) and BREG (P = 0.0001) at month 8 compared with baseline. CONCLUSION: As compared with healthy controls, patients with PMN displayed a lower percentage of BREGs. Both TREGs and BREGs significantly improved with disease-specific therapy. BREGs had an association with clinical activity.

Exploring Cadaver Skin For Standardization Of Rabbit And Porcine Burn Models In Research.

Burn animal models provide substantial insights into burn pathophysiology. Choice of the apt model is important for determining the clinical efficacy of new medicines. Therefore, standardization of burn models is crucial for scientific research. Use of common techniques like hot water, electricity and incandescent instruments to generate animal burn models is widely reported. However, great discrepancy in employed temperature and exposure times demands user-dependent standardization of the animal model prior to research. Establishment of custom generated in vivo burn models giving consideration to reduced use, suffering and risk of the experimental animal is equally crucial. Accordingly, this pilot study demonstrates a novel approach using rabbit and porcine cadaver skin for standardization of burn parameters prior to use in live animal models. Using a custom-made soldering iron coupled to a 16cm2 surface area copper plate, burns at randomly chosen temperatures of 80˚C and 120˚C, with exposure times ranging from 60s to 180s, were produced on rabbit and porcine cadaver skins. On gross and histopathological analysis, parameters required to generate characteristic changes for deep partial and full thickness burn involvement were established. The identified temperature and exposure time parameters were further validated in live animal models. In vivo validation established the success of this approach, highlighting reduced animal use, ease, reproducibility and efficacy in burn model standardization. The findings of this study will hopefully encourage researchers to opt for cadaver skin to determine parameters required to generate a specific degree of burn prior to its use in live animals for burn research.

Les modèles animaux permettent des avancées substantielles dans la compréhension de la physiopathologie des brûlures. Le choix du modèle est important pour évaluer l'efficacité de nouvelles thérapeutiques, donc la standardisation de ces modèles est cruciale. Les utilisations de l'eau chaude, de l'électricité et de solides chauds est couramment décrite. Cependant, la grande variabilité des températures et des temps de contact nécessite une calibration avant chaque étude expérimentale. Il est en outre nécessaire de développer des modèles in vivo à façon tout en réduisant le risque d'erreur et la souffrance animale. Cette étude décrit l'utilisation de peaux de lapins et de porcs morts pour la standardisation des modèles de brûlure les utilisant. Nous utilisons un fer à souder couplé à une plaque de cuivre de 16 cm², pouvant délivrer une température réglable entre 80 et 120°C pendant 60 à 180 s sur de la peau de lapin ou de porc morts. Nous avons défini les paramètres permettant de réaliser des brûlures intermédiaires ou profondes, objectivées par études macro- et microscopiques, validés ensuite chez l'animal vivant. Cette étape a permis de confirmer l'efficacité de cette approche qui réduit le nombre d'animaux d'expérience, est aisée et reproductible et bien corrélée à l'approche in vivo. Cette étude devrait conduire les équipes a réaliser une calibration ex vivo avant de réaliser une étude in vivo de brûlure expérimentale.

Fever of Unknown Origin in a Renal Transplant Recipient: Lactate Dehydrogenase as an Important Clue to Diagnosis.

Histoplasmosis is a rare disease in nonendemic areas. We report a case of a 23-year-old male patient who presented with fever of unknown origin, cytopenias, organomegaly, and allograft dysfunction 4 months after renal transplant with father as donor. Bone marrow examination showed intracellular budding yeast cells, which was confirmed as histoplasmosis by culture of bone marrow biopsy sample. The patient was treated with intravenous liposomal amphotericin and responded well.

Development of a recombinant murine tumour model using hepatoma cells expressing hepatitis C virus nonstructural antigens.

Hepatitis C virus (HCV) chronically infects 2%-3% of the world's population, causing liver disease and cancer with prolonged infection. The narrow host range of the virus, being restricted largely to human hepatocytes, has made the development of relevant models to evaluate the efficacy of vaccines a challenge. We have developed a novel approach to accomplish this by generating a murine hepatoma cell line stably expressing nonstructural HCV antigens which can be used in vitro or in vivo to test HCV vaccine efficacies. These HCV-recombinant hepatoma cells formed large solid-mass tumours when implanted into syngeneic mice, allowing us to test candidate HCV vaccines to demonstrate the development of an HCV-specific immune response that limited tumour growth. Using this model, we tested the therapeutic potential of recombinant anti-HCV-specific vaccines based on two fundamentally different attenuated pathogen vaccine systems-attenuated Salmonella and recombinant adenoviral vector based vaccine. While attenuated Salmonella that secreted HCV antigens limited growth of the HCV-recombinant tumours when used in a therapeutic vaccination trial, replication-competent but noninfectious adenovirus expressing nonstructural HCV antigens showed overall greater survival and reduced weight loss compared to non-replicating nondisseminating adenovirus. Our results demonstrate a model with anti-tumour responses to HCV nonstructural (NS) protein antigens and suggest that recombinant vaccine vectors should be explored as a therapeutic strategy for controlling HCV and HCV-associated cancers.

Superior immunogenicity of HCV envelope glycoproteins when adjuvanted with cyclic-di-AMP, a STING activator or archaeosomes.

Three decades after the discovery, hepatitis C virus (HCV) is still the leading cause of liver transplantation and poses a major threat to global health. In spite of recent advances in the development of direct acting antivirals, there is still a need for a prophylactic vaccine to limit the virus spread and protect at-risk populations, especially in developing countries, where the cost of the new treatments may severely limit access. The use of recombinant HCV glycoproteins E1E2 (rE1E2) in combination with the MF59, an oil-in-water emulsion-based adjuvant, has previously been shown to reduce the rate of chronicity in chimpanzees and to induce production of cross-neutralizing antibodies and cellular immune responses in human volunteers. To further improve neutralizing antibody responses in recipients along with robust T cell responses, we have explored the immunogenicity of different adjuvants when formulated with the HCV rE1E2 vaccine in mice. Our data show that cyclic di-adenosine monophosphate (c-di-AMP) and archaeosomes elicit strong neutralizing antibodies similar to those elicited using aluminum hydroxide/monophosphoryl lipid A (Alum/monophos. /MPLA) and MF59. However, both c-di-AMP and archaeosomes induced a more robust cellular immune response, which was confirmed by the detection of vaccine-specific poly-functional CD4+ T cells. We conclude that these adjuvants may substantially boost the immunogenicity of our E1E2 vaccine. In addition, our data also indicates that use of a partial or exclusive intranasal immunization regimen may also be feasible using c-di-AMP as adjuvant.

Archaeal lipid vaccine adjuvants for induction of cell-mediated immunity.

INTRODUCTION: Liposomal vesicles (archaeosomes) composed of total polar lipids (TPL) or semi-synthetic glycerolipids, unique to the domain Archaea, constitute potent vaccine adjuvant and delivery systems. The characteristics of this adjuvant offer a novel prospect for the development of effective vaccines for emerging infections and cancers, which is reviewed in this article. Areas covered: The areas covered in this review include the chemical composition and physical characteristics, various in-vitro and in-vivo pre-clinical immunogenicity and efficacy studies for systemic immunization, induction of mucosal immunity upon modification of the formulation with cations, and the mechanism of adjuvant action following uptake by antigen presenting cells. Expert commentary: The unique features of archaeal lipids confer archaeosomes with many desirable features. With the use of semi-synthetic archaeosomes, highly defined lipids that are safe and robust for induction of cell-mediated immunity may be chosen. These adjuvants function as Toll-like receptor-independent innate immune stimulants.

Generation of a functional liver tissue mimic using adipose stromal vascular fraction cell-derived vasculatures.

One of the major challenges in cell implantation therapies is to promote integration of the microcirculation between the implanted cells and the host. We used adipose-derived stromal vascular fraction (SVF) cells to vascularize a human liver cell (HepG2) implant. We hypothesized that the SVF cells would form a functional microcirculation via vascular assembly and inosculation with the host vasculature. Initially, we assessed the extent and character of neovasculatures formed by freshly isolated and cultured SVF cells and found that freshly isolated cells have a higher vascularization potential. Generation of a 3D implant containing fresh SVF and HepG2 cells formed a tissue in which HepG2 cells were entwined with a network of microvessels. Implanted HepG2 cells sequestered labeled LDL delivered by systemic intravascular injection only in SVF-vascularized implants demonstrating that SVF cell-derived vasculatures can effectively integrate with host vessels and interface with parenchymal cells to form a functional tissue mimic.

IL-10 produced by trophoblast cells inhibits phagosome maturation leading to profound intracellular proliferation of Salmonella enterica Typhimurium.

INTRODUCTION: Salmonella enterica Typhimurium (ST) is a phagosomal pathogen that can infect placental trophoblast cells leading to abortion and severe maternal illness. It is unclear how the trophoblast cells promote profound bacterial proliferation. METHODS: The mechanism of internalization, intracellular growth and phagosomal biogenesis in ST-infected human epithelial (HeLa), macrophage (THP-1) and trophoblast-derived cell lines (JEG-3, BeWo and HTR-8) was studied. Specific inhibitors were used to block bacterial internalization. Phagosomal maturation was determined by confocal microscopy, Western-blotting and release of lysosomal ß-galactosidase by infected cells. Bacterial colony forming units were determined by plating infected cell lysates on agar plates. RESULTS: ST proliferated minimally in macrophages but replicated profoundly within trophoblast cells. The ST-ΔinvA (a mutant of Salmonella pathogenicity island-1 gene effector proteins) was unable to infect epithelial cells, but was internalized by scavenger receptors on trophoblasts and macrophages. However, ST was contrastingly localized in early (Rab5⁺) or late (LAMP1⁺) phagosomes within trophoblast cells and macrophages respectively. Furthermore trophoblast cells (unlike macrophages) did not exhibit phagoso-lysosomal fusion. ST-infected macrophages produced IL-6 whereas trophoblast cells produced IL-10. Neutralizing IL-10 in JEG-3 cells accelerated phagolysomal fusion and reduced proliferation of ST. Placental bacterial burden was curtailed in vivo in anti-IL-10 antibody treated and IL-10-deficient mice. DISCUSSION: Macrophages phagocytose but curtail intracellular replication of ST in late phagosomes. In contrast, phagocytosis by trophoblast cells results in an inappropriate cytokine response and proliferation of ST in early phagosomes. CONCLUSION: IL-10 production by trophoblast cells that delays phagosomal maturation may facilitate proliferation of pathogens in placental cells.

cIAP1 and cIAP2 limit macrophage necroptosis by inhibiting Rip1 and Rip3 activation.

Cellular inhibitor of apoptosis proteins (cIAPs) have emerged as important anti-cell death mediators, particularly in cancer. Although they are known to be expressed in immune tissue, their specific immune function remains unclear. We observed that degradation of cIAPs with SMAC mimetic (SM) results in death of primary bone-marrow-derived macrophages. SM-induced death of macrophages occurred by programmed necrosis (necroptosis), which was dependent on TNF receptor expression. Consistent with necroptosis, SM-induced death of macrophages was abrogated by inhibition of receptor interacting protein 1 (Rip1) kinase signaling or by receptor interacting protein 3 (Rip3) knockdown. SM-induced necroptosis was also dependent on inhibition of SM-induced apoptosis due to the expression of the endogenous caspase inhibitor, xIAP. We found that cIAPs limit Rip3, and to a lesser extent Rip1, expression via post-transcriptional mechanisms, leading to inhibition of the Rip1-Rip3 death complex (necrosome). Reduced cIAP activity in vivo, via SM treatment or specific knockout of either cIAP, resulted in elevated macrophage cell death and compromised control of an intracellular bacterium, Listeria monocytogenes. These results show that cIAPs have an important role in limiting programmed necrosis of macrophages, which facilitates effective control of a pathogen.

Large gastric perforation in carmi syndrome: a morbid complication in a rare association.

The association between epidermolysis bullosa (EB) and congenital pyloric atresia (CPA) named Carmi Syndrome is rare. We report unusual and morbid complication of gastric perforation resulting in peritonitis in a preterm neonate born with Carmi Syndrome.

Cellular immunoisolation for islet transplantation by a novel dual porosity electrospun membrane.

Immunoisolation strategies have the potential to impact the treatment of several diseases, such as hemophilia, Parkinson's and endocrine disorders, such as parathryroid disorders and diabetes. The hallmark of these disease states is the amelioration of the disease process by replacement of the deficient protein. Naturally, several cellular therapeutic strategies like genetically modified host cells, stem cells, donor cells, or even complex tissues like pancreatic islets have been investigated. Current evidence suggests that successful strategies must incorporate considerations for local hypoxia, vascularity, and immunoisolation. Additional regulatory concerns also include safe localization of implanted therapeutic cells to allow for monitoring, dose adjustment, or removal when indicated. Local hypoxia and cellular toxicity can be detrimental to the survival of freshly implanted pancreatic islets, leading to a need for a larger initial number of islets or repeated implantation procedures. The lack of adequate donors and the large number of islet equivalents needed to achieve euglycemic states amplify the nature of this problem. We have developed a novel immunoisolation device based on electrospun nylon, primarily for islet transplantation, such that the inner component functions as a cellular barrier while allowing diffusion, whereas the outer component can be optimized for tissue integration and accelerated vascularization. Devices explanted after subcutaneous implantation in wild-type B6 mice after a period of 30 days show vascular elements in the outer layer of the electrospun device. The inner layer when intact functioned as an effective barrier to cellular infiltration. The preimplantation of such a device, with a relatively thin inner barrier membrane, will allow for adequate vascularization and reduce postimplantation hypoxia. This study demonstrates the feasibility of an electrospun isolation device that can be easily assembled, modified by varying the electrospinning parameters, and functionalized with surface-active molecules to accelerate vascularization.

Adult stem cell homing and differentiation in vitro on composite fibrin matrix.

Strategies to generate differentiated cells from haematopoetic progenitor cells will enhance potential use of adult stem cells for therapeutic transplantation or tissue engineering. Transplantation of undifferentiated stem cells into recipient tissue hinges on the hypothesis of a milieu dependent differentiation and it has been suggested that a clot-equivalent scaffold is crucial for these circulating cells to anchor and multiply. Here a natural scaffold, fibrin along with fibronectin, gelatin and growth factors has been used to induce endothelial progenitor cells and smooth muscle progenitor cells to differentiate into endothelial cells and smooth muscle cells, respectively, from peripheral blood mononuclear cells. Characteristics of endothelial cells have been verified by the detection of mRNA for and immunostaining the cells for von Willebrand factor, uptake of acetylated low-density lipoproteins and measurement of released nitric oxide in the culture medium, as nitrite. The specific molecules that characterized smooth muscle cells were alpha smooth muscle actin and calponin, besides deposition of collagen type I and elastin, onto the culture matrix. The adhesive proteins used for the fabrication of endothelial progenitor cells matrix and smooth muscle progenitor cells matrix were the same, but specific differentiation was brought about by modulating the growth factor composition in the matrix and in the culture medium. Both endothelial and smooth muscle cells were consistently developed from 20 ml of human blood.

A comparison of neural feature extraction methods for brain-machine interfaces.

Brain-machine interferes (BMIs) have shown promise in augmenting people's control of their surroundings, especially for those suffering from paralysis due to neurological disorders. This paper describes an experiment using the rodent model to explore information available in neural signals recorded from chronically implanted intracortical microelectrode arrays. In offline experiments, a number of neural feature extraction methods were utilized to obtain neural activity vectors (NAVs) describing the activity of the underlying neural population while rats performed a discrimination task. The methods evaluated included standard techniques such as binned spike rates and local field potential spectra as well as more novel approaches including matched-filter energy, raw signal spectra, and an autocorrelation energy measure (AEM) approach. Support vector machines (SVMs) were trained offline to classify left from right going movements by utilizing features contained in the NAVs obtained by the different methods. Each method was evaluated for accuracy and robustness. Results show that most algorithms worked well for decoding neural signals both during and prior to movement, with spectral methods providing the best stability.

Liposome adjuvants prepared from the total polar lipids of Haloferax volcanii, Planococcus spp. and Bacillus firmus differ in ability to elicit and sustain immune responses.

Immune stimulating activity was compared for lipid vesicles consisting of the total polar lipids of an archaeon Haloferax volcanii, and the eubacteria Planococcus spp. and Bacillus firmus. Each total polar lipid extract readily formed liposomes of similar size, within which the protein antigen ovalbumin was entrapped, with comparable loading and internalization. Subcutaneous immunization of mice resulted in anti-ovalbumin antibody titers for all adjuvants, with memory recall responses that were significantly greater with the archaeal lipid (H. volcanii versus Planococcus). More striking, induction of cytotoxic T cell activity against the entrapped antigen, measured 10 days following a single vaccination (primary response) rapidly declined by week 7 (secondary response after injections on days 0 and 21) in mice immunized with Planococcus spp. liposomes, but was sustained in mice immunized with H. volcanii archaeosomes. Surprisingly, antigen free-Planococcus liposomes evoked potent non-specific inflammatory cytokine production (IL-12 and IL-6) by dendritic cells whereas archaeal H. volcanii vesicles evoked little inflammatory cytokines. This suggested that overt inflammatory response might not necessarily aid sustenance of immunity. B. firmus liposomes consisted of phosphatidylglycerol, phosphatidylethanolamine and cardiolipin and was an ineffective CTL adjuvant, even for initiating a primary response. Considering that the polar lipids of H. volcanii and Planococcus spp. both consist of the same lipid classes (sulfoglycolipids, phosphoglycerols, and cardiolipins), the unique ability of archaeosomes to maintain antigen-specific T cell immunity may be attributable to a property of the archaeal 2,3-diphytanylglycerol lipid core.

Protease action and generation of beta-thromboglobulin-like protein followed by platelet activation.

Beta-thromboglobulin (beta-TG) is a platelet-specific protein present in the alpha-granules and secreted into the surrounding medium on cell activation. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of platelet releasate after inhibition of metalloproteinases with ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetra acetic acid (EGTA) showed disappearance of an 8.0-kDa band. In the absence of the cation chelators, a 48-kDa band disappeared and concurrently, the 8.0-kDa band intensity increased suggesting that the former may be the immediate precursor of the latter. The Western blot stained using specific antibodies, isolated from single-cell clones of hybridoma, against 8.0 kDa protein recognized not only 48 and 8.0 kDa bands but few others too. The data suggests that one or more high molecular weight protein is released from alpha-granules and is broken down into smaller fragments after release to form beta-thromboglobulin (beta-TG)-like proteins by the action of metal dependent proteases.

Quantitation of platelet adhesion to Ti and DLC-coated Ti in vitro using (125)I-labeled platelets.

Measurement of platelet adhesion in vitro is a good indicator of its reactivity to implant devices in vivo. Platelets were labeled with I-125 without affecting its normal morphology and function and the labeled platelets were suspended in platelet poor plasma and exposed to Ti and diamond like carbon-coated (DLC) Ti discs, under dynamic conditions, using a parallel plate flow chamber. The test materials were washed, dried, exposed to a phosphor screen and scanned to get the images. The number of platelets that adhered to Ti was found to be higher than those that adhered to DLC coated Ti sample, irrespective of the shear stress which was varied between 2 and 16 dynes/cm(2).

Apolipoprotein C3 SstI polymorphism and triglyceride levels in Asian Indians.

BACKGROUND: A close association between Sst I polymorphism in the 3' untranslated region of the apolipoproteinC3 (APOC3) gene and levels of plasma triglycerides (TG) had been reported by different investigators. Hypertriglyceridemia(HTG) is a known risk factor for coronary artery disease (CAD) in the context of Asian Indians. We conducted a study on the relationship between APOC3 SstI polymorphism (S1S1, S1S2 and S2S2 genotypes) and plasma TG levels in a group of 139 male healthy volunteers from Northern India. METHODS: DNA samples were analyzed by polymerase chain reaction (PCR) followed by SstI digestion. Digested PCR products were run on 3% agarose gel and visualized by ethidium bromide staining. RESULTS: Rare S2 allele was highly prevalent in our study population (0.313) as compared to the Caucasians (0.00-0.11). The genotypic distribution was in agreement with Hardy-Weinberg equilibrium. S2 allele was almost two times more prevalent in the HTG group (N = 34) as compared to NTG group (N = 105) (p = 0.001). Multiple logistic regression revealed S1S2 individuals had age-adjusted odds ratio of 2.43 (95%CI = 0.99-6.01, p = 0.054) and S2S2 had 9.9 (95%CI = 2.66-37.29, p = 0.0006) for developing HTG in comparison to S1S1 genotype. CONCLUSIONS: Our study shows a significant association between rare S2 allele and HTG in Asian Indians.