Spleen Tyrosine Kinase phosphorylates VE-cadherin to cause endothelial barrier disruption in acute lung injury.

Increased endothelial cell (EC) permeability is a cardinal feature of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Tyrosine phosphorylation of VE-cadherin is a key determinant of EC barrier disruption. However, the identity and role of tyrosine kinases in this context are incompletely understood. Here we report that Spleen Tyrosine Kinase (Syk) is a key mediator of EC barrier disruption and lung vascular leak in sepsis. Inhibition of Syk by pharmacological or genetic approaches, each reduced thrombin-induced EC permeability. Mechanistically, Syk associates with and phosphorylates VE-cadherin to cause EC permeability. To study the causal role of endothelial Syk in sepsis-induced ALI, we used a remarkably efficient and cost-effective approach based on gene transfer to generate EC-ablated Syk mice. These mice were protected against sepsis-induced loss of VE-cadherin and inflammatory lung injury. Notably, the administration of Syk inhibitor R788 (fostamatinib); currently in phase II clinical trial for the treatment of COVID-19, mitigated lung injury and mortality in mice with sepsis. These data identify Syk as a novel kinase for VE-cadherin and a druggable target against ALI in sepsis.

The Protective Role of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Protein Sigma-1 Receptor in Regulating Endothelial Inflammation and Permeability Associated with Acute Lung Injury.

Earlier studies from our lab identified endoplasmic reticulum (ER) chaperone BiP/GRP78, an important component of MAM, to be a novel determinant of endothelial cell (EC) dysfunction associated with acute lung injury (ALI). Sigma1R (Sig1R) is another unique ER receptor chaperone that has been identified to associate with BiP/GRP78 at the MAM and is known to be a pluripotent modulator of cellular homeostasis. However, it is unclear if Sig1R also plays a role in regulating the EC inflammation and permeability associated with ALI. Our data using human pulmonary artery endothelial cells (HPAECs) showed that siRNA-mediated knockdown of Sig1R potentiated LPS-induced the expression of proinflammatory molecules ICAM-1, VCAM-1 and IL-8. Consistent with this, Sig1R agonist, PRE-084, known to activate Sig1R by inducing its dissociation from BiP/GRP78, blunted the above response. Notably, PRE-084 failed to blunt LPS-induced inflammatory responses in Sig1R-depleted cells, confirming that the effect of PRE-084 is driven by Sig1R. Furthermore, Sig1R antagonist, NE-100, known to inactivate Sig1R by blocking its dissociation from BiP/GRP78, failed to block LPS-induced inflammatory responses, establishing that dissociation from BiP/GRP78 is required for Sig1R to exert its anti-inflammatory action. Unlike Sig1R, the siRNA-mediated knockdown or Subtilase AB-mediated inactivation of BiP/GRP78 protected against LPS-induced EC inflammation. Interestingly, the protective effect of BiP/GRP78 knockdown or inactivation was abolished in cells that were depleted of Sig1R, confirming that BiP/GRP78 knockdown/inactivation-mediated suppression of EC inflammation is mediated via Sig1R. In view of these findings, we determined the in vivo relevance of Sig1R in a mouse model of sepsis-induced ALI. The intraperitoneal injection of PRE-084 mitigated sepsis-induced ALI, as evidenced by a decrease in ICAM-1, IL-6 levels, lung PMN infiltration, and lung vascular leakage. Together, these data evidence a protective role of Sig1R against endothelial dysfunction associated with ALI and identify it as a viable target in terms of controlling ALI in sepsis.

4 Dimensional XStrain speckle tracking echocardiography: comprehensive evaluation of left ventricular strain and twist parameters in healthy Indian adults during COVID-19 pandemic.

INTRODUCTION: 4D XStrain speckle tracking echocardiography (STE) is a feasible newer technology to evaluate the strain and rotational deformation of left ventricle (LV). We aimed to exhaustively present the normal value ranges of LV strain and twist parameter in healthy Indian adults during COVID-19 pandemic and furthermore to analyse their relationship with age and gender. METHOD: Study population consisted of 80 adults of 18-60 years (58 men, 22 women), which was arbitrarily divided into two groups: Group A <30 years and Group B >31 years. RESULTS: GLS was higher in females (P<0.01) and in Group A (P<0.01). On the contrary GCS and GRS were higher in men (P=NS) and in Group B (P<0.01), at the mitral valve level. At the papillary muscle level GCS and GRS values are more in men (P<0.01) and in <30 years of age (P<0.01 and P<0.05 respectively). Furthermore, the values of numerous other strain parameters-GLSR, GCSR, GRSR, LGV, TV, TS, TSR, Shear, Shear rate, ROV and RV, reflected heterogeneous variation across gender and various age groups. Twist was greater in men and increased with increasing age (P<0.01). CONCLUSION: We have demonstrated a comprehensive data obtained in the current study utilizing 4D XStrain STE in healthy subjects. The LV speckle tracking software simultaneously provided 4D volumetric, strain, rotation and twist data in great detail. However, this distinctive technology has not been widely adopted and its evaluation is still limited to research applications. Therefore, further clinical studies are needed to validate our findings.

Autophagy protein ATG7 is a critical regulator of endothelial cell inflammation and permeability.

Endothelial cell (EC) inflammation and permeability are critical pathogenic mechanisms in many inflammatory conditions including acute lung injury. In this study, we investigated the role of ATG7, an essential autophagy regulator with no autophagy-unrelated functions, in the mechanism of EC inflammation and permeability. Knockdown of ATG7 using si-RNA significantly attenuated thrombin-induced expression of proinflammatory molecules such as IL-6, MCP-1, ICAM-1 and VCAM-1. Mechanistic study implicated reduced NF-κB activity in the inhibition of EC inflammation in ATG7-silenced cells. Moreover, depletion of ATG7 markedly reduced the binding of RelA/p65 to DNA in the nucleus. Surprisingly, the thrombin-induced degradation of IκBα in the cytosol was not affected in ATG7-depleted cells, suggesting a defect in the translocation of released RelA/p65 to the nucleus in these cells. This is likely due to suppression of thrombin-induced phosphorylation and thereby inactivation of Cofilin1, an actin-depolymerizing protein, in ATG7-depleted cells. Actin stress fiber dynamics are required for thrombin-induced translocation of RelA/p65 to the nucleus, and indeed our results showed that ATG7 silencing inhibited this response via inactivation of Cofilin1. ATG7 silencing also reduced thrombin-mediated EC permeability by inhibiting the disassembly of VE-cadherin at adherens junctions. Together, these data uncover a novel function of ATG7 in mediating EC inflammation and permeability, and provide a mechanistic basis for the linkage between autophagy and EC dysfunction.

Seed interior microbiome of rice genotypes indigenous to three agroecosystems of Indo-Burma biodiversity hotspot.

BACKGROUND: Seeds of plants are a confirmation of their next generation and come associated with a unique microbia community. Vertical transmission of this microbiota signifies the importance of these organisms for a healthy seedling and thus a healthier next generation for both symbionts. Seed endophytic bacterial community composition is guided by plant genotype and many environmental factors. In north-east India, within a narrow geographical region, several indigenous rice genotypes are cultivated across broad agroecosystems having standing water in fields ranging from 0-2 m during their peak growth stage. Here we tried to trap the effect of rice genotypes and agroecosystems where they are cultivated on the rice seed microbiota. We used culturable and metagenomics approaches to explore the seed endophytic bacterial diversity of seven rice genotypes (8 replicate hills) grown across three agroecosystems. RESULTS: From seven growth media, 16 different species of culturable EB were isolated. A predictive metabolic pathway analysis of the EB showed the presence of many plant growth promoting traits such as siroheme synthesis, nitrate reduction, phosphate acquisition, etc. Vitamin B12 biosynthesis restricted to bacteria and archaea; pathways were also detected in the EB of two landraces. Analysis of 522,134 filtered metagenomic sequencing reads obtained from seed samples (n=56) gave 4061 OTUs. Alpha diversity indices showed significant differences in observed OTU richness (P≤0.05) across genotypes. Significant differences were also found between the individual hills of a rice genotype. PCoA analysis exhibited three separate clusters and revealed the clusters separated based on genotype, while agroecosystem showed a minimal effect on the variation of seed microbiota (adonis, R2=0.07, P=0.024). Interestingly, animal gut resident bacteria such as Bifidobacterium, Faecalibacterium, Lactobacillus, etc. were found in abundance as members of the seed microbiota. CONCLUSION: Overall, our study demonstrates, indigenous rice genotypes of north-east India have a unique blend of endophytic bacteria in their mature seeds. While there are notable variations among plants of the same genotype, we found similarities among genotypes cultivated in completely different environmental conditions. The beta diversity variations across the seven rice genotypes were significantly shaped by their genotype rather than their agroecosystems.

Corrigendum: EBI-3 Chain of IL-35 Along With TGF-ß Synergistically Regulate Anti-leishmanial Immunity.

[This corrects the article DOI: 10.3389/fimmu.2019.00616.].

A Novel Marine Natural Product Derived Pyrroloiminoquinone with Potent Activity against Skin Cancer Cells.

Non-melanoma skin cancer is one of the major ailments in the United States. Effective drugs that can cure skin cancers are limited. Moreover, the available drugs have toxic side effects. Therefore, skin cancer drugs with less toxic side effects are urgently needed. To achieve this goal, we focused our work on identifying potent lead compounds from marine natural products. Five lead compounds identified from a class of pyrroloiminoquinone natural products were evaluated for their ability to selectively kill squamous cell carcinoma (SCC13) skin cancer cells using an MTT assay. The toxicity of these compounds was also evaluated against the normal human keratinocyte HaCaT cell line. The most potent compound identified from these studies, C278 was further evaluated for its ability to inhibit cancer cell migration and invasion using a wound-healing assay and a trans-well migration assay, respectively. To investigate the molecular mechanism of cell death, the expression of apoptotic and autophagy proteins was studied in C278 treated cells compared to untreated cells using western blot. Our results showed that all five compounds effectively killed the SCC13 cells, with compound C278 being the most effective. Compound C278 was more effective in killing the SCC13 cells compared to HaCaT cells with a two-fold selectivity. The migration and the invasion of the SCC13 cells were also inhibited upon treatment with compound C278. The expression of pro-apoptotic and autophagy proteins with concomitant downregulation in the expression of survival proteins were observed in C278 treated cells. In summary, the marine natural product analog compound C278 showed promising anticancer activity against human skin cancer cells and holds potential to be developed as an effective anticancer agent to combat skin cancer.

EB1-3 Chain of IL-35 Along With TGF-ß Synergistically Regulate Anti-leishmanial Immunity.

Immunosuppression is a characteristic feature of chronic leishmaniasis. The dynamicity and the functional cross talks of host immune responses during Leishmania infection are still not clearly understood. Here we explored the functional aspects of accumulation of immune suppressive cellular and cytokine milieu during the progression of murine visceral leishmaniasis. In addition to IL-10 and TGF-ß, investigation on the responses of different subunit chains of IL-12 family revealed a progressive elevation of EBI-3 and p35 chains of EBI-3 with Leishmania donovani infection in BALB/c mice. The expansion of CD25 and FoxP3 positive T cells is associated with loss of IFN-γ and TNF-α response in advanced disease. Ex-vivo and in vivo neutralization of TGF-ß and EBI-3 suggests a synergism in suppression of host anti-leishmanial immunity. The down-regulation of EBI-3 and TGF-ß is crucial for re-activation of JAK-STAT pathway for induction as well as restoration of protective immunity against L. donovani infection.

RNA-Seq Revealed Expression of Many Novel Genes Associated With Leishmania donovani Persistence and Clearance in the Host Macrophage.

Host- as well as parasite-specific factors are equally crucial in allowing either the Leishmania parasites to dominate, or host macrophages to resist infection. To identify such factors, we infected murine peritoneal macrophages with either the virulent (vAG83) or the non-virulent (nvAG83) parasites of L. donovani. Then, through dual RNA-seq, we simultaneously elucidated the transcriptomic changes occurring both in the host and the parasites. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed (DE) genes, we showed that the vAG83-infected macrophages exhibit biased anti-inflammatory responses compared to the macrophages infected with the nvAG83. Moreover, the vAG83-infected macrophages displayed suppression of many important cellular processes, including protein synthesis. Further, through protein-protein interaction study, we showed significant downregulation in the expression of many hubs and hub-bottleneck genes in macrophages infected with vAG83 as compared to nvAG83. Cell signaling study showed that these two parasites activated the MAPK and PI3K-AKT signaling pathways differentially in the host cells. Through gene ontology analyses of the parasite-specific genes, we discovered that the genes for virulent factors and parasite survival were significantly upregulated in the intracellular amastigotes of vAG83. In contrast, genes involved in the immune stimulations, and those involved in negative regulation of the cell cycle and transcriptional regulation, were upregulated in the nvAG83. Collectively, these results depicted a differential regulation in the host and the parasite-specific molecules during in vitro persistence and clearance of the parasites.

Genome Plasticity in Cultured Leishmania donovani: Comparison of Early and Late Passages.

Leishmania donovani possesses a complex heteroxenic life cycle where infective metacyclic promastigotes are pre-adapted to infect their host and cope up with intracellular stress. Exploiting the similarities between cultured and sandfly derived promastigotes, we used early and late passage cultured promastigotes to show specific changes at genome level which compromise pathogen fitness reflected in gene expression and infection studies. The pathogen loses virulence mostly via transcriptional and translational regulations and long-time cultivation makes them struggle to convert to virulent metacyclics. At the genomic level very subtle plasticity was observed between the early and the late passages mostly in defense-related, nutrient acquisition and signal transduction genes. Chromosome Copy number variation is seen in the early and late passages involving several genes that may be playing a role in pathogenicity. Our study highlights the importance of ABC transporters and calpain like cysteine proteases in parasite virulence in cultured promastigotes. Interestingly, these proteins are emerging as important patho-adaptive factors in clinical isolates of Leishmania. We found that the currently available genome of Leishmania in the NCBI database are from late passages. Our early passage genome can act as a reference for future studies on virulent isolates of Leishmania. The annotated leads from this study can be used for virulence surveillance and therapeutic studies in the Indian subcontinent.

Fracture of mandible during yawning in a patient with osteogenesis imperfecta.

Osteogenesis imperfecta is a genetic disorder characterised by fragility and multiple fractures of bones. Clinical signs and symptoms vary depending on the type of disease. Fractures of facial bones are rare compared with load-bearing long bones. We report a case of fracture of the mandible during yawning which was managed by open reduction and internal fixation.

Occupational Health Hazards among Sewage Workers: Oxidative Stress and Deranged Lung Functions.

BACKGROUND: Sewage workers, because of their occupation, are exposed to different types of dusts, bio-aerosols, fumes and gases like methane, hydrogen sulfide, sulphur dioxide, etc, which contribute towards oxidative stress and detrimental effects on various body functions, especially lung functions. AIMS AND OBJECTIVES: This study was carried out on sewage workers (who had been working for more than five years). We wanted to study the role of oxidative stress in development of impaired lung functions among sewage workers. MATERIALS AND METHODS: This cross sectional study was done in a tertiary care hospital (J.N. Medical College) in Aligarh, U.P. Study was done from March 2008 to December 2009. The study group comprised of 62 sewage workers who had been working for more than five years (32 non-smokers and 30 smokers) and 60 control subjects (30 smokers and 30 non-smokers). The pulmonary functions of these workers were assessed by using a MIR (Medical International Lab) Spiro Lab II Spirometer, with subjects in sitting position. Valid written consents were obtained from all the subjects. Malondialdehyde (MDA) is produced as a result of the action of reactive oxygen species (ROS) on the lipids present in the membranes of the cells, especially, contracting muscle cells. Serum MDA levels were assessed as an indirect measure of oxidative stress in these sewage workers and they were compared with serum MDA levels of control subjects. Appropriate statistical tests were applied for analysis of the data which was generated. OBSERVATION AND RESULTS: There were statistically significant decreases in Peak Expiratory Flow Rate (PEFR), Forced Expiratory Volume in first second (FEV1) and FEV1/FVC percent ratio (<80%) and Forced Expiratory Flow at 25%-75% of volume as percentage of Vital Capacity (FEF 25%-75%). Also, we found statistically significant increased levels of serum MDA in these sewage workers as compared to those in control subjects (with a p-value of <0.05 with a confidence interval of 95%). CONCLUSION: Our study found that the occupational exposure of the sewage workers to harmful dust, fumes, gases and bio-aerosols contributed to oxidative stress among them. This oxidative stress was one of the mechanisms which led to the development of obstructive impairment of lung functions in these sewage workers.

Evaluation of soft and hard tissue changes after bimaxillary surgery in class III orthognathic surgery and aesthetic consideration.

AIMS: The aim of this study was to evaluate hard and soft tissue change after bimaxillary surgery in class III patients by focusing on sella, nasion, A point (SNA) and sella, nasion, B point (SNB) angle and aesthetic outcome. MATERIALS AND METHODS: The sample consisted of 96 skeletal Class III patients (42 women, 54 men) with a mean age of 25 years with standard deviation (SD) of 8.4. The youngest patient was 16-years-old and the oldest 51-years-old at the time of surgery. In total, seven skeletal parameters, eight soft tissue parameters, and two dental parameters were evaluated on the cephalograms. RESULT: At the beginning of the treatment 49 Patients had SNA between 80° and 84°, 34 had SNA of less than 80° and 13 had SNA of more than 84°. Post surgically, 25 patients had SNA of 78°-84°, 19 had SNA less than 78° and 52 patients had SNA of more than 84°. Out of 96 patients 22 had SNB of 78°-82° before surgery, 16 had less than 78° and 58 had SNA of more than 84°. Postoperatively, we measured SNB of 78°-80° in 42, less than 78° in 18 and of more than 82° in 36 patients. The inclination of the maxilla relative to the cranial base changed from 7.2° (SD = 4)-8° (SD = 5.1) and the mandible changed from 35.7° (SD = 6.6) to 36° (SD = 6.3) postoperatively which was not significant. The distance from upper lip to E-line increased by 2.6 mm (SD = 3.9) after surgery (P < 0.001), while, the lower lip distance to E-line decreased slightly by 0.9 mm (SD = 3.2) (P < 0.01). Nasolabial angle was decreased by 9.5° (SD = 9.4) after surgery (P < 0.001). The nose prominence also decreased from 18.2 mm (SD = 3.5) -16.5 mm (SD = 3.3). CONCLUSION: Although in many cases we did not have a SNA angle or SNB angle in normal range but a good aesthetic outcome have been observed. Consequently our study showed that soft tissue change and aesthetic aspects should be considered in surgical planning and achieving SNA angle or SNB angle of norm range should not be the only goal. As we could show the advancement of maxilla will result in a better lip and nose profile and this should be considered in treatment planning.

Oral metastases from carcinoma of cervix.

Metastatic tumours of the oral cavity are uncommon, they may occur in soft tissue as well as in bone in the oropharyngeal region. Owing to its rarity, metastatic tumours of the oral regions are a challenge to diagnose. We report a case of metastasis of the oral cavity, arising from uterine cervix mimicking as mucoepidermoid carcinoma. The metastatic lesions were noticed in the soft tissue of the lower buccal and gingival side of a oral cavity, in a 40-year-old woman with history of an adenosquamous carcinoma of uterine cervix treated by panhysterectomy.