Piezo1 mechanosensing regulates integrin-dependent chemotactic migration in human T cells.

T cells are crucial for efficient antigen-specific immune responses and thus their migration within the body, to inflamed tissues from circulating blood or to secondary lymphoid organs, plays a very critical role. T cell extravasation in inflamed tissues depends on chemotactic cues and interaction between endothelial adhesion molecules and cellular integrins. A migrating T cell is expected to sense diverse external and membrane-intrinsic mechano-physical cues, but molecular mechanisms of such mechanosensing in cell migration are not established. We explored if the professional mechanosensor Piezo1 plays any role during integrin-dependent chemotaxis of human T cells. We found that deficiency of Piezo1 in human T cells interfered with integrin-dependent cellular motility on ICAM-1-coated surface. Piezo1 recruitment at the leading edge of moving T cells is dependent on and follows focal adhesion formation at the leading edge and local increase in membrane tension upon chemokine receptor activation. Piezo1 recruitment and activation, followed by calcium influx and calpain activation, in turn, are crucial for the integrin LFA1 (CD11a/CD18) recruitment at the leading edge of the chemotactic human T cells. Thus, we find that Piezo1 activation in response to local mechanical cues constitutes a membrane-intrinsic component of the 'outside-in' signaling in human T cells, migrating in response to chemokines, that mediates integrin recruitment to the leading edge.

Shifts in Pseudomonas species diversity influence adaptation of brown planthopper to changing climates and geographical locations.

The brown planthopper (BPH) is a monophagous sap-sucking pest of rice that causes immense yield loss. The rapid build-up of pesticide resistance combined with the ability of BPH populations to quickly overcome host plant resistance has rendered conventional control strategies ineffective. One of the likely ways in which BPH adapts to novel environments is by undergoing rapid shifts in its microbiome composition. To elucidate the rapid adaptation to novel environments and the contributions of Pseudomonas toward insect survival, we performed Pseudomonas-specific 16S rRNA gut-microbiome profiling of BPH populations. Results revealed the differential occurrence of Pseudomonas species in BPH populations with changing climates and geographical locations. Further, the observed variation in Pseudomonas species composition and abundance correlated with BPH survivability. Collectively, this study, while adding to our current understanding of symbiont-mediated insect adaptation, also demonstrated a complex interplay between insect physiology and microbiome dynamics, which likely confers BPH its rapid adaptive capacity.

Delineating the complex mechanistic interplay between NF-κß driven mTOR depedent autophagy and monocyte to macrophage differentiation: A functional perspective.

Autophagy is an extremely essential cellular process aimed to clear redundant and damaged materials, namely organelles, protein aggregates, invading pathogens, etc. through the formation of autophagosomes which are ultimately targeted to lysosomal degradation. In this study, we demonstrated that mTOR dependent classical autophagy is ubiquitously triggered in differentiating monocytes. Moreover, autophagy plays a decisive role in sustaining the process of monocyte to macrophage differentiation. We have delved deeper into understanding the underlying mechanistic complexities that trigger autophagy during differentiation. Intrigued by the significant difference between the protein profiles of monocytes and macrophages, we investigated to learn that autophagy directs monocyte differentiation via protein degradation. Further, we delineated the complex cross-talk between autophagy and cell-cycle arrest in differentiating monocytes. This study also inspects the contribution of adhesion on various steps of autophagy and its ultimate impact on monocyte differentiation. Our study reveals new mechanistic insights into the process of autophagy associated with monocyte differentiation and would undoubtedly help to understand the intricacies of the process better for the effective design of therapeutics as autophagy and autophagy-related processes have enormous importance in human patho-physiology.

MAP Kinase driven actomyosin rearrangement is a crucial regulator of monocyte to macrophage differentiation.

Rearrangement of actin cytoskeleton correlates significantly with the immune responses as the perturbation of cytoskeletal dynamics leads to many immune deficiencies. Mechanistic insights into this correlation remain unknown. Cellular spreading, the most characteristic phenotype associated with monocyte to macrophage differentiation, led us to investigate the contribution of actomyosin dynamics in monocyte differentiation. Our observation revealed that actomyosin reorganization intrinsically governs the process of monocyte to macrophage differentiation. Further, we established that the MAPK-driven signaling pathways regulate the cellular actomyosin dynamics that direct monocyte to macrophage differentiation. We also identified P42/44 Mitogen-Activated Protein Kinase (P42/44 MAPK), P38 Mitogen-Activated Protein Kinase (P38 MAPK), MAP Kinase Activated Protein Kinase 2 (MK-2), Heat Shock Protein 27 (Hsp-27), Lim Kinase (Lim K), non-muscle cofilin (n-cofilin), Myosin Light Chain Kinase (MLCK) and Myosin Light Chain (MLC) as critical components of the signaling network. Moreover, we have shown the involvement of the same signaling cascade in 3D gel-like microenvironment induced spontaneous monocyte to macrophage differentiation and in human blood-derived PBMC differentiation. Our study reveals new mechanistic insights into the process of monocyte to macrophage differentiation.

Reactive oxygen species-mediated cytoplasmic stiffening impairs the phagocytic ability of the macrophage.

The phagocytic ability of macrophages empowers them to enforce innate immunity. RAW264.7, THP-1 and peripheral blood mononuclear cell-derived macrophages display considerable variability with regards to their phagocytic ability. We identify the underlying causes that attenuate the phagocytic abilities of a macrophage. Deformability of the cytoplasm and cortex influences the macrophage's phagocytic ability, and macrophages use the large cell-to-cell variability of their cytoplasmic stiffness to modulate their phagocytic ability. We find that the more-deformable macrophages have a higher phagocytic ability than those that are less deformable. Further, the subcellular spatial variability of cortex stiffness gives rise to more-deformable subdomains on the membrane for pathogen ingestion. We report a previously unknown negative-feedback loop that is triggered by the phagocytic oxidative burst. Macrophages utilize the excess reactive oxygen species to stiffen the cytoplasm, reducing their phagocytic propensity. In organisms, ageing or pathological conditions impair the phagocytic ability of macrophages. Our findings identify the targets that could potentially be utilized for restoring the phagocytic ability of the defunct macrophages.

Molecular characterization and functional analysis of the vitellogenin receptor from eri silkworm, Samia ricini.

The vitellogenin receptor (VgR) plays a critical role in egg development by mediating endocytosis of the major yolk protein precursor vitellogenin (Vg). Therefore, identifying the VgR of beneficial insects and its characterization could lead to the development of novel egg production strategies to enhance their commercial values. Here, we present the cloning, expression, and functional characterization of the VgR from an economically important eri silkworm, Samia ricini. The complete mRNA sequence was 6002 bp with an ORF of 5484 bp, encoding a protein of 1827 amino acids. Sequence analyses revealed that the SrVgR contained all of the conservative structural motifs characteristics of LDLR family members. The SrVgR was specifically expressed in the ovary, and the mRNA level increased steadily in pupal stages, reached its peak on day 9, and then declined to a bare minimum in adults. RNA interference (RNAi) clearly reduced the VgR transcript levels, disrupted the ovarian development resulting in malformed ovarioles and abnormal development of eggs. Taken together, these data provide conclusive evidence for the essential roles of VgR in insect reproduction.

Environmental and Molecular Drivers of the α-Gal Syndrome.

The α-Gal syndrome (AGS) is a type of allergy characterized by an IgE antibody (Ab) response against the carbohydrate Galα1-3Galß1-4GlcNAc-R (α-Gal), which is present in glycoproteins from tick saliva and tissues of non-catarrhine mammals. Recurrent tick bites induce high levels of anti-α-Gal IgE Abs that mediate delayed hypersensitivity to consumed red meat products in humans. This was the first evidence that tick glycoproteins play a major role in allergy development with the potential to cause fatal delayed anaphylaxis to α-Gal-containing foods and drugs and immediate anaphylaxis to tick bites. Initially, it was thought that the origin of tick-derived α-Gal was either residual blood meal mammalian glycoproteins containing α-Gal or tick gut bacteria producing this glycan. However, recently tick galactosyltransferases were shown to be involved in α-Gal synthesis with a role in tick and tick-borne pathogen life cycles. The tick-borne pathogen Anaplasma phagocytophilum increases the level of tick α-Gal, which potentially increases the risk of developing AGS after a bite by a pathogen-infected tick. Two mechanisms might explain the production of anti-α-Gal IgE Abs after tick bites. The first mechanism proposes that the α-Gal antigen on tick salivary proteins is presented to antigen-presenting cells and B-lymphocytes in the context of Th2 cell-mediated immunity induced by tick saliva. The second mechanism is based on the possibility that tick salivary prostaglandin E2 triggers Immunoglobulin class switching to anti-α-Gal IgE-producing B cells from preexisting mature B cells clones producing anti-α-Gal IgM and/or IgG. Importantly, blood group antigens influence the capacity of the immune system to produce anti-α-Gal Abs which in turn impacts individual susceptibility to AGS. The presence of blood type B reduces the capacity of the immune system to produce anti-α-Gal Abs, presumably due to tolerance to α-Gal, which is very similar in structure to blood group B antigen. Therefore, individuals with blood group B and reduced levels of anti-α-Gal Abs have lower risk to develop AGS. Specific immunity to tick α-Gal is linked to host immunity to tick bites. Basophil activation and release of histamine have been implicated in IgE-mediated acquired protective immunity to tick infestations and chronic itch. Basophil reactivity was also found to be higher in patients with AGS when compared to asymptomatic α-Gal sensitized individuals. In addition, host resistance to tick infestation is associated with resistance to tick-borne pathogen infection. Anti-α-Gal IgM and IgG Abs protect humans against vector-borne pathogens and blood group B individuals seem to be more susceptible to vector-borne diseases. The link between blood groups and anti-α-Gal immunity which in turn affects resistance to vector-borne pathogens and susceptibility to AGS, suggests a trade-off between susceptibility to AGS and protection to some infectious diseases. The understanding of the environmental and molecular drivers of the immune mechanisms involved in AGS is essential to developing tools for the diagnosis, control, and prevention of this growing health problem.

Self-Assembling Peptide-Based Hydrogel: Regulation of Mechanical Stiffness and Thermal Stability and 3D Cell Culture of Fibroblasts.

A histidine-containing peptide-based amphiphile (P1) forms a transparent hydrogel within a pH range of 5.5 to 8.5 in phosphate buffer solution. Interestingly, thermal stability and mechanical stiffness are modulated by incorporating different types of dicarboxylic acids into the hydrogels. Inclusion of succinic acid with the molar ratio 2:1 (peptide:dicarboxylic acid) yields improved properties compared to the other tested dicarboxylic acids such as oxalic, glutaric and octanedioic acids. Transmission electron microscopic (TEM) images show the assembly of nanospheres is responsible for the hydrogel obtained from the assembly of native peptide. However, a morphological transformation takes place from nanosphere to nanofibers, when the peptide gels with succinic acid. XRD and FT-IR studies reveal interactions between peptide amphiphiles and the acids are responsible for the formation of a two-component hydrogel. Gel stiffness is enhanced considerably upon the addition of succinic acid to P1 with a 1:2 molar ratio. The two-component gel consisting of peptide and succinic acid has been successfully used for three-dimensional cell culture using mouse fibroblast cell line (NIH-3T3). This indicates future promise for the application of such peptide-based gels as tunable biomaterials in cell culture and regenerative medicine.

3D micro-environment regulates NF-κß dependent adhesion to induce monocyte differentiation.

Differentiation of monocytes entails their relocation from blood to the tissue, hence accompanied by an altered physicochemical micro-environment. While the mechanism by which the biochemical make-up of the micro-environment induces differentiation is known, the fluid-like to gel-like transition in the physical micro-environment is not well understood. Monocytes maintain non-adherent state to prevent differentiation. We establish that irrespective of the chemical makeup, a 3D gel-like micro-environment induces a positive-feedback loop of adhesion-MAPK-NF-κß activation to facilitate differentiation. In 2D fluid-like micro-environment, adhesion alone is capable of inducing differentiation via the same positive-feedback signaling. Chemical inducer treatment in fluid-like micro-environment, increases the propensity of monocyte adhesion via a brief pulse of p-MAPK. The adhesion subsequently elicit differentiation, establishing that adhesion is both necessary and sufficient to induce differentiation in 2D/3D micro-environment. MAPK, and NF-κß being key molecules of multiple signaling pathways, we hypothesize that biochemically inert 3D gel-like micro-environment would also influence other cellular functions.

Lipid chain saturation and the cholesterol in the phospholipid membrane affect the spectroscopic properties of lipophilic dye nile red.

We have studied the effect of composition and the phase state of phospholipid membranes on the emission spectrum, anisotropy and lifetime of a lipophilic fluorescence probe nile red. Fluorescence spectrum of nile red in membranes containing cholesterol has also been investigated in order to get insights into the influence of cholesterol on the phospholipid membranes. Maximum emission wavelength (λem) of nile red in the fluid phase of saturated and unsaturated phospholipids was found to differ by ~10nm. The λem was also found to be independent of chain length and charge of the membrane. However, the λem is strongly dependent on the temperature in the gel phase. The λem and rotational diffusion rate decrease, whereas the anisotropy and lifetime increase markedly with increasing cholesterol concentration for saturated phosoholipids, such as, dimyristoyl phosphatidylcholine (DMPC) in the liquid ordered phase. However, these spectroscopic properties do not alter significantly in case of unsaturated phospholipids, such as, dioleoyl phosphatidylcholine (DOPC) in liquid disordered phase. Interestingly, red edge excitation shift (REES) in the presence of lipid-cholesterol membranes is the direct consequences of change in rotational diffusion due to motional restriction of lipids in the presence of cholesterol. This study provides correlations between the membrane compositions and fluorescence spectral features which can be utilized in a wide range of biophysical fields as well the cell biology.

Bacterial Community Structure in the Asian Rice Gall Midge Reveals a Varied Microbiome Rich in Proteobacteria.

The Asian rice gall midge (ARGM) has emerged as a model gall forming pest of rice. The ARGM infestation of rice results in failure of panicle formation and economic loss. Understanding the molecular basis of ARGM-rice interactions is very crucial in order to control this devastating pest of rice. The current investigation was devised to identify bacterial communities present in the ARGM and in addition the bacterial diversity in the maggots during their interaction with susceptible or resistant rice varieties. Sequencing of 16S rRNA bacterial gene (V3-V4 region) revealed differences in the microflora of the ARGM maggots feeding on susceptible or resistant rice hosts. Results revealed that Wolbachia was the predominant bacterium in pupae and adults while Pseudomonas was predominant in maggots. Further, we observed that members of proteobacteria were predominant across all the samples. There was high species diversity in maggots isolated from susceptible rice and a high representation of unclassified bacteria in maggots isolated from resistant rice. This is the first study that reports variation of microbiome of the ARGM, based on host phenotype from which it was isolated, and results suggest that these variations could have an important role in host's susceptibility.

Zebrafish lipid droplets regulate embryonic ATP homeostasis to power early development.

In zebrafish embryos, the maternally supplied pool of ATP is insufficient to power even the earliest of developmental events (0-3 hpf) such as oocyte-to-embryo transition (OET). The embryos generate an additional pulse (2.5 h long) of ATP (1.25-4 hpf) to achieve the embryonic ATP homeostasis. We demonstrate that the additional pulse of ATP is needed for successful execution of OET. The maternally supplied yolk lipids play a crucial role in maintaining the embryonic ATP homeostasis. In this paper, we identify the source and trafficking routes of free fatty acids (FFAs) that feed the mitochondria for synthesis of ATP. Interestingly, neither the maternally supplied pool of yolk-FFA nor the yolk-FACoA (fatty acyl coenzyme A) is used for ATP homeostasis during 0-5 hpf in zebrafish embryos. With the help of lipidomics, we explore the link between lipid droplet (LD)-mediated lipolysis and ATP homeostasis in zebrafish embryos. Until 5 hpf, the embryonic LDs undergo extensive lipolysis that generates FFAs. We demonstrate that these newly synthesized FFAs from LDs are involved in the maintenance of embryonic ATP homeostasis, rather than the FFAs/FACoA present in the yolk. Thus, the LDs are vital embryonic organelles that maintain the ATP homeostasis during early developmental stages (0-5 hpf) in zebrafish embryos. Our study highlights the important roles carried on by the LDs during the early development of the zebrafish embryos.

Genomics of the Asian rice gall midge and its interactions with rice.

Understanding virulence and manipulative strategies of gall formers will reveal new facets of plant defense and insect counter defense. Among the gall midges, the Asian rice gall midge (AGM) has emerged as a model for studies on plant-insect interactions. Data from several genomics, transcriptomics and metabolomics studies have revealed diverse strategies adopted by AGM to successfully invade the host while overcoming its defense. Adaptive skills of AGM transcend from its genomic and transcriptomic make-up. Information arising from studies on genetics, mitochondrial genome and miRNAs, amongst other parameters, highlights AGM's capacity to maneuver the host defense, reorient host metabolome and redirect its morphogenesis.

Differential Gene Expression of BRCA1,ERBB2 and TP53 biomarkers between Human Breast Tissue and Peripheral Blood Samples of Breast Cancer.

Breast cancer is a most common malignancy especially in Iraqi women accounting for high morbidity and mortality. Mutations in BRCA1 gene is one of the important genetic predisposing factors inbreast cancer. Similarly ERBB2 and TP53 are also key prognostic markers in breast cancer treatment.We were interested to explore the gene expression profiles of BRCA1, ERBB2 and TP53 in breast cancer women patients from Iraq so as to assess the potential of such markers in breast cancer treatment. The mRNA levels were significantly over-expressed in tumor tissues in comparison to normal ones with p values (p<0.005) observed between malignant BRCA1 and control tissue samples. Similarly significant difference (p<0.001) was observed between malignant ERBB2 in comparison to control, and malignant TP53 and benign tissue samples as well. However in blood samples, no considerable expression of these markers was observed. Out of three selected genes, ERBB2 expression was significantly expressed in comparison to BRCA1 and TP53 in cancer tissue. Mutation analysis of BRCA1, ERBB2 and TP53 has been made to find out the region most susceptible to mutations in these genes The BRCA1 exon 11, ERBB2 16 and TP53 exon 5 displayed increased chances of having mutations. We can conclude from the study that differential gene expression of BRCA1, ERBB2 and TP53 at mRNA levels may act as a diagnostic marker of circulating tumor cells having important prognostic value in breast cancer patients.

Pyrosequencing-based transcriptome analysis of the asian rice gall midge reveals differential response during compatible and incompatible interaction.

The Asian rice gall midge (Orseolia oryzae) is a major pest responsible for immense loss in rice productivity. Currently, very little knowledge exists with regard to this insect at the molecular level. The present study was initiated with the aim of developing molecular resources as well as identifying alterations at the transcriptome level in the gall midge maggots that are in a compatible (SH) or in an incompatible interaction (RH) with their rice host. Roche 454 pyrosequencing strategy was used to develop both transcriptomics and genomics resources that led to the identification of 79,028 and 85,395 EST sequences from gall midge biotype 4 (GMB4) maggots feeding on a susceptible and resistant rice variety, TN1 (SH) and Suraksha (RH), respectively. Comparative transcriptome analysis of the maggots in SH and RH revealed over-representation of transcripts from proteolysis and protein phosphorylation in maggots from RH. In contrast, over-representation of transcripts for translation, regulation of transcription and transcripts involved in electron transport chain were observed in maggots from SH. This investigation, besides unveiling various mechanisms underlying insect-plant interactions, will also lead to a better understanding of strategies adopted by insects in general, and the Asian rice gall midge in particular, to overcome host defense.

Serine proteases-like genes in the asian rice gall midge show differential expression in compatible and incompatible interactions with rice.

The Asian rice gall midge, Orseolia oryzae (Wood-Mason), is a serious pest of rice. Investigations into the gall midge-rice interaction will unveil the underlying molecular mechanisms which, in turn, can be used as a tool to assist in developing suitable integrated pest management strategies. The insect gut is known to be involved in various physiological and biological processes including digestion, detoxification and interaction with the host. We have cloned and identified two genes, OoprotI and OoprotII, homologous to serine proteases with the conserved His(87), Asp(136) and Ser(241) residues. OoProtI shared 52.26% identity with mosquito-type trypsin from Hessian fly whereas OoProtII showed 52.49% identity to complement component activated C1s from the Hessian fly. Quantitative real time PCR analysis revealed that both the genes were significantly upregulated in larvae feeding on resistant cultivar than in those feeding on susceptible cultivar. These results provide an opportunity to understand the gut physiology of the insect under compatible or incompatible interactions with the host. Phylogenetic analysis grouped these genes in the clade containing proteases of phytophagous insects away from hematophagous insects.

Isolation and characterization of microsatellite loci in the Asian rice gall midge (Orseolia oryzae) (Diptera: Cecidomyiidae).

Microsatellite loci were isolated from the genomic DNA of the Asian rice gall midge, Orseolia oryzae (Wood-Mason) using a hybridization capture approach. A total of 90 non-redundant primer pairs, representing unique loci, were designed. These simple sequence repeat (SSR) markers represented di (72%), tri (15.3%), and complex repeats (12.7%). Three biotypes of gall midge (20 individuals for each biotype) were screened using these SSRs. The results revealed that 15 loci were hyper variable and showed polymorphism among different biotypes of this pest. The number of alleles ranged from two to 11 and expected heterozygosity was above 0.5. Inheritance studies with three markers (observed to be polymorphic between sexes) revealed sex linked inheritance of two SSRs (Oosat55 and Oosat59) and autosomal inheritance of one marker (Oosat43). These markers will prove to be a useful tool to devise strategies for integrated pest management and in the study of biotype evolution in this important rice pest.

Dynamic organization of chromatin assembly and transcription factories in living cells.

The interphase nucleus is an active organelle involved in processing genetic information. In higher order eukaryotes, information control is compartmentalized - for example at the scale of inter-chromosome territories and nuclear bodies. Regulatory proteins, nuclear bodies and chromatin assembly are found to be highly dynamic within the nucleus of primary cells and through cellular differentiation programs. In this chapter we describe live-cell fluorescence based techniques and single particle tracking analysis, to probe the spatio-temporal dimension in nuclear function.

Photoactivation of the CreER T2 recombinase for conditional site-specific recombination with high spatiotemporal resolution.

We implemented a noninvasive optical method for the fast control of Cre recombinase in single cells of a live zebrafish embryo. Optical uncaging of the caged precursor of a nonendogeneous steroid by one- or two-photon illumination was used to restore Cre activity of the CreER(T2) fusion protein in specific target cells. This method labels single cells irreversibly by inducing recombination in an appropriate reporter transgenic animal and thereby can achieve high spatiotemporal resolution in the control of gene expression. This technique could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration, or carcinogenesis) with high spatiotemporal resolution (single cell and 10-min scales).

Photocontrol of protein activity in cultured cells and zebrafish with one- and two-photon illumination.

We have implemented a noninvasive optical method for the fast control of protein activity in a live zebrafish embryo. It relies on releasing a protein fused to a modified estrogen receptor ligand binding domain from its complex with cytoplasmic chaperones, upon the local photoactivation of a nonendogenous caged inducer. Molecular dynamics simulations were used to design cyclofen-OH, a photochemically stable inducer of the receptor specific for 4-hydroxy-tamoxifen (ER(T2)). Cyclofen-OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ER(T2) receptor. This was shown in cultured cells and in zebrafish embryos through emission properties and subcellular localization of properly engineered fluorescent proteins. Cyclofen-OH was successfully caged with various photolabile protecting groups. One particular caged compound was efficient in photoinducing the nuclear translocation of fluorescent proteins either globally (with 365 nm UV illumination) or locally (with a focused UV laser or with two-photon illumination at 750 nm). The present method for photocontrol of protein activity could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration and carcinogenesis) with high spatiotemporal resolution.