Distinct developmental pathways generate functionally distinct populations of natural killer cells.

Natural killer (NK) cells function by eliminating virus-infected or tumor cells. Here we identified an NK-lineage-biased progenitor population, referred to as early NK progenitors (ENKPs), which developed into NK cells independently of common precursors for innate lymphoid cells (ILCPs). ENKP-derived NK cells (ENKP_NK cells) and ILCP-derived NK cells (ILCP_NK cells) were transcriptionally different. We devised combinations of surface markers that identified highly enriched ENKP_NK and ILCP_NK cell populations in wild-type mice. Furthermore, Ly49H+ NK cells that responded to mouse cytomegalovirus infection primarily developed from ENKPs, whereas ILCP_NK cells were better IFNγ producers after infection with Salmonella and herpes simplex virus. Human CD56dim and CD56bright NK cells were transcriptionally similar to ENKP_NK cells and ILCP_NK cells, respectively. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets in mice and further suggest these pathways may be conserved in humans.

Modulation of the tumor microenvironment and mechanism of immunotherapy-based drug resistance in breast cancer.

Breast cancer, the most frequent female malignancy, is often curable when detected at an early stage. The treatment of metastatic breast cancer is more challenging and may be unresponsive to conventional therapy. Immunotherapy is crucial for treating metastatic breast cancer, but its resistance is a major limitation. The tumor microenvironment (TME) is vital in modulating the immunotherapy response. Various tumor microenvironmental components, such as cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs), are involved in TME modulation to cause immunotherapy resistance. This review highlights the role of stromal cells in modulating the breast tumor microenvironment, including the involvement of CAF-TAM interaction, alteration of tumor metabolism leading to immunotherapy failure, and other latest strategies, including high throughput genomic screening, single-cell and spatial omics techniques for identifying tumor immune genes regulating immunotherapy response. This review emphasizes the therapeutic approach to overcome breast cancer immune resistance through CAF reprogramming, modulation of TAM polarization, tumor metabolism, and genomic alterations.

Comparison of inflammatory markers in low-pressure pneumoperitoneum with deep neuromuscular block versus standard pressure pneumoperitoneum among patients undergoing laparoscopic cholecystectomy for gallstone disease: a randomized control trial.

BACKGROUND: Low-pressure pneumoperitoneum (LPP) is an attempt to improve laparoscopic surgery. Lower pressure causes lesser inflammation and better hemodynamics. There is a lack of literature comparing inflammatory markers in LPP with deep NMB to standard pressure pneumoperitoneum (SPP) with moderate NMB in laparoscopic cholecystectomy. METHODOLOGY: This was a single institutional prospective randomized control trial. Participants included all patients undergoing laparoscopic cholecystectomy for symptomatic gall stone disease. Participants were divided into 2 groups group A and B. Group A-Low-pressure group in which pneumoperitoneum pressure was kept low (8-10 mmHg) with deep Neuromuscular blockade (NMB) and Group B-Normal pressure group (12-14 mmHg) with moderate NMB. A convenience sample size of 80 with 40 in each group was selected. Lab investigations like CBC, LFT, RFT and serum IL-1, IL-6, IL-17, TNF alpha levels were measured at base line and 24 h after surgery and compared using appropriate statistical tests. Other parameters like length of hospital stay, post-operative pain score, conversion rate (low-pressure to standard pressure), and complications were also compared. RESULTS: Eighty participants were analysed with 40 in each group. Baseline characteristics and investigations were statistically similar. Difference (post-operative-pre-operative) of inflammatory markers were compared between both groups. Numerically there was a slightly higher rise in most of the inflammatory markers (TLC, ESR, CRP, IL-6, TNFα) in Group B compared to Group A but not statistically significant. Albumin showed significant fall (p < 0.001) in Group B compared to Group A. Post-operative pain was also significantly less (p < 0.001) in Group A compared to Group B at 6 h and 24 h. There were no differences in length of hospital stay and incidence of complications. There was no conversion from low-pressure to standard pressure. CONCLUSION: Laparoscopic cholecystectomy performed under low-pressure pneumoperitoneum with deep NMB may have lesser inflammation and lesser post-operative pain compared to standard pressure pneumoperitoneum with moderate NMB. Future studies with larger sample size need to be designed to support these findings.

The ChaC family of γ-glutamyl cyclotransferases is required for Leishmania to switch to a slow growth state and for long-term survival of the parasite.

The ChaC family of γ-glutamyl cyclotransferases is conserved throughout all Kingdoms and catalyzes the degradation of GSH. So far, the ChaC family proteins in trypanosomal parasites are missing in the literature. Here, we report two members of the ChaC family of γ-glutamyl cyclotransferases (LmChaC2a and LmChaC2b) in the unicellular pathogen Leishmania. Activity measurements suggest that these proteins catalyze degradation of GSH but no other γ-glutamyl peptides. Recombinant LmChaC2a protein shows ∼17-fold lower catalytic efficiency (kcat ∼ 0.9 s-1) than LmChaC2b (kcat ∼ 15 s-1), although they showed comparable Km values (∼1.75 mM for LmChaC2a and ∼2.0 mM for LmChaC2b) toward GSH. qRT-PCR and Western blot analyses suggest that the LmChaC2a protein was found to be constitutively expressed, whereas LmChaC2b was regulated by sulfur stress. To investigate its precise physiological function in Leishmania, we generated overexpressed, knockout, and complement cell lines. Flow cytometric analyses show the presence of a higher intracellular GSH concentration and lower intracellular ROS level, indicative of a more reductive environment in null mutants. We found LmChaC2-expressing cells grow in GSH-containing sulfur-limited media, while the null mutants failed to grow, suggesting that LmChaC2 is crucial for cell growth with GSH as the only sulfur source. Null mutants, although reach the stationary phase rapidly, display impaired long-term survival, indicating that LmChaC2-mediated GSH degradation is necessary for prolonged survival. In vivo studies suggest that LmChaC2-dependent controlled GSH degradation promotes chronic infection by the parasite. Altogether, these data indicate that LmChaC2 plays an important role in GSH homeostasis in Leishmania.

CRISPR based therapeutics: a new paradigm in cancer precision medicine.

BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems are the latest addition to the plethora of gene-editing tools. These systems have been repurposed from their natural counterparts by means of both guide RNA and Cas nuclease engineering. These RNA-guided systems offer greater programmability and multiplexing capacity than previous generation gene editing tools based on zinc finger nucleases and transcription activator like effector nucleases. CRISPR-Cas systems show great promise for individualization of cancer precision medicine. MAIN BODY: The biology of Cas nucleases and dead Cas based systems relevant for in vivo gene therapy applications has been discussed. The CRISPR knockout, CRISPR activation and CRISPR interference based genetic screens which offer opportunity to assess functions of thousands of genes in massively parallel assays have been also highlighted. Single and combinatorial gene knockout screens lead to identification of drug targets and synthetic lethal genetic interactions across different cancer phenotypes. There are different viral and non-viral (nanoformulation based) modalities that can carry CRISPR-Cas components to different target organs in vivo. CONCLUSION: The latest developments in the field in terms of optimization of performance of the CRISPR-Cas elements should fuel greater application of the latter in the realm of precision medicine. Lastly, how the already available knowledge can help in furtherance of use of CRISPR based tools in personalized medicine has been discussed.

Variations within Toll-like receptor (TLR) and TLR signaling pathway-related genes and their synergistic effects on the risk of Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is the commonest post-infectious polyradiculopathy. Although genetic background of the host seems to play an important role in the susceptibility to GBS, genes conferring major risk are not yet known. Dysregulation of Toll-like receptor (TLR) molecules exacerbates immune-inflammatory responses and the genetic variations within TLR pathway-related genes contribute to differential risk to infection. The aim of this study was to delineate the impact of genetic variations within TLR2, TLR3, and TLR4 genes as well as TLR signaling pathway-related genes such as MyD88, TRIF, TRAF3, TRAF6, IRF3, NFκß1, and IκBα on risk of developing GBS. Fourteen polymorphisms located within TLR2 (rs3804099, rs111200466), TLR3 (rs3775290, rs3775291), TLR4 (rs1927911, rs11536891), MyD88 (rs7744, rs4988453), TRIF (rs8120), TRAF3 (rs12147254), TRAF6 (rs4755453), IRF3 (rs2304204), NFκß1 (rs28362491), and IκBα (rs696) genes were genotyped in 150 GBS patients and 150 healthy subjects either by PCR-RFLP or TaqMan Allelic Discrimination Assay. Genotypes of two polymorphic variants, Del/Del of rs111200466 insertion and deletion (INDEL) polymorphism of TLR2 gene and TT of rs3775290 single nucleotide polymorphism (SNP) of TLR3 gene had significantly higher frequencies among GBS patients, while the frequencies of TT genotype of rs3804099 SNP of TLR2 gene and TT genotype of rs11536891 SNP of TLR4 gene were significantly higher in controls. Gene-gene interaction study by Multifactor Dimensionality Reduction analysis also suggested a significant combined effect of TLR2, and NFκß1 genes on the risk of GBS. The SNPs in the IκBα and IRF3 genes correlated with severity of GBS. The genes encoding TLRs and TLR signaling pathway-related molecules could serve as crucial genetic markers of susceptibility and severity of GBS.

Straightforward Access to Anthrone Functionalized Benzylic Amines via Organocatalytic 1,2-Addition of Anthrones to Imines at Ambient Temperature.

Activation of anthrone via benzylic deprotonation in the presence of triethylamine paves the way for the 1,2-addition reaction with imines to provide the desired functionalized anthrones in good to excellent yields under mild and operationally simple reaction conditions with a broad range of substrate scopes without using any external additives or toxic stoichiometric reagents.

Antecedent infections in Guillain-Barré syndrome patients from south India.

Guillain-Barré syndrome (GBS) is the commonest post-infectious inflammatory peripheral neuropathy with undiscerned aetiology. The commonly reported antecedent infections implicated in India include Campylobacter jejuni, chikungunya, dengue, and Japanese encephalitis (JE). In this study from south India, we investigated the role of these four agents in triggering GBS. This case-control study was performed on 150 treatment-naive patients with GBS and 150 age and sex-matched controls from the same community. IgM immunoreactivity for C. jejuni, chikungunya, and dengue was detected by enzyme-linked immunosorbent assay (ELISA) in serum of patients with GBS and control subjects. Immunoreactivity against JE was detected in serum as well as cerebrospinal fluid (CSF) from patients (n = 150) and orthopaedic control (n = 45) subjects. The immunoreactivity against infections was compared between demyelinating and axonal subtypes of GBS. Overall, 119/150 patients with GBS had serological evidence of antecedent infection. Amongst those with evidence of antecedent infection, 24 (16%), 8 (5%), and 9 (6%) patients were exclusively immunoreactive to chikungunya, JE, and C. jejuni, respectively. In the remaining patients (78/119), immunoreactivity to multiple pathogens was noted. Immunoreactivity to C. jejuni infection was found in 32% of GBS patients compared to 2.7% controls (P < .001), whereas to chikungunya virus was reported in 66.7% of patients with GBS compared to 44.7% controls (P = .006). Anti-dengue immunoreactivity was significantly associated with the demyelinating subtype of GBS. Patients positive for JE IgM (CSF) manifested demyelinating electrophysiology. In this large case-control study, immunoreactivity against multiple infectious agents was observed in a subset of patients. Chikungunya was the commonest antecedent infection, followed by C. jejuni.

Iridium-catalyzed direct C-H arylation of cyclic N-sulfonyl ketimines with arylsiloxanes at ambient temperature.

An iridium-catalyzed ortho-selective C-H arylation of cyclic N-sulfonyl ketimines has been achieved with environmentally benign aryl siloxanes. The reaction is highly efficient and proceeds at ambient temperature which is the key feature of the methodology considering the weak coordination nature of the substrate as well as the sluggish reactivity of siloxanes. A wide array of pharmaceutically relevant novel biaryls has been synthesized under operationally simple conditions.

Deciphering the near-field response with the far-field wavelength-scanned SERS spectra of 4-mercaptopyridine adsorbed on gold nanocolloidal particles entrapped in Langmuir Reverse Schaefer film of 5CB liquid crystal molecules.

Herein, we have reported the wavelength-scanned (WS) SERS spectra of 4-mercaptopyridine molecules (4-MPy) adsorbed on gold nanoparticles immobilised in a Langmuir Reverse Schaefer (L-RSh) film matrix of 5CB molecules. The WS-SERS spectral profile exhibited an increment in the intensity of the enhanced Raman bands of 4-MPy with an increase in the wavelength of the excitation laser source. The rationale behind the experimental observations was supported by the simulated extinction spectra and the enhancement factor measurements of the modelled systems using the T-matrix formalism. The SERS intensity fluctuations in the band located at 1000 cm-1 for the 4-MPy molecule, as obtained from three different locations in the L-RSh film substrate, were also analyzed. Surprisingly, depending on the spatial locations of the substrates, the intensity fluctuations of the abovementioned band exhibit both Poisson and Gaussian distributions but the maximum number of probe molecules that can reside in the scattering areas under investigation cannot exceed sixteen. These observations suggest that the origin of SERS from single/few molecules or from the ensemble-averaged system cannot be inferred from the statistical distributions of the Raman intensity fluctuations. The present experimental observations also revealed the relation between the near-field plasmonic behaviors of the substrate with the corresponding far-field SERS spectra of the 4-MPy molecule.

Multiple recurrences of spindle cell oncocytoma: A case report and literature review.

Spindle cell oncocytoma (SCO) is a rare tumor originating from pituicytes of the neurohypophysis. It typically occurs in the adult population between 50 and 60 years of age and is often misdiagnosed preoperatively as pituitary adenoma because of similar clinical presentation and neuroimaging features. Diffuse nuclear immunoreactivity for TTF-1 (also known as NK2 homeobox 1 factor) is characteristic of SCO and other tumors of the posterior pituitary. We report a case of SCO in a patient who underwent multiple surgical resections for recurrence. The aim of this case report and literature review is to provide an overview of what is currently known about SCO as well as raise the awareness of this entity to endocrinologists, neurologists, neurosurgeons, and neuropathologists. Our case is also unusual in that the tumor displayed immunoreactivity for neuronal markers, which is a very rare occurrence.

PAS domain-containing phosphoglycerate kinase deficiency in Leishmania major results in increased autophagosome formation and cell death.

Per-Arnt-Sim (PAS) domains are structurally conserved and present in numerous proteins throughout all branches of the phylogenetic tree. Although PAS domain-containing proteins are major players for the adaptation to environmental stimuli in both prokaryotic and eukaryotic organisms, these types of proteins are still uncharacterized in the trypanosomatid parasites, Trypanosome and Leishmania In addition, PAS-containing phosphoglycerate kinase (PGK) protein is uncharacterized in the literature. Here, we report a PAS domain-containing PGK (LmPAS-PGK) in the unicellular pathogen Leishmania The modeled structure of N-terminal of this protein exhibits four antiparallel ß sheets centrally flanked by α helices, which is similar to the characteristic signature of PAS domain. Activity measurements suggest that acidic pH can directly stimulate PGK activity. Localization studies demonstrate that the protein is highly enriched in the glycosome and its presence can also be seen in the lysosome. Gene knockout, overexpression and complement studies suggest that LmPAS-PGK plays a fundamental role in cell survival through autophagy. Furthermore, the knockout cells display a marked decrease in virulence when host macrophage and BALB/c mice were infected with them. Our work begins to clarify how acidic pH-dependent ATP generation by PGK is likely to function in cellular adaptability of Leishmania.

Acceptability of Using a Robotic Nursing Assistant in Health Care Environments: Experimental Pilot Study.

BACKGROUND: According to the US Bureau of Labor Statistics, nurses will be the largest labor pool in the United States by 2022, and more than 1.1 million nursing positions have to be filled by then in order to avoid a nursing shortage. In addition, the incidence rate of musculoskeletal disorders in nurses is above average in comparison with other occupations. Robot-assisted health care has the potential to alleviate the nursing shortage by automating mundane and routine nursing tasks. Furthermore, robots in health care environments may assist with safe patient mobility and handling and may thereby reduce the likelihood of musculoskeletal disorders. OBJECTIVE: This pilot study investigates the perceived ease of use and perceived usefulness (acceptability) of a customized service robot as determined by nursing students (as proxies for nursing staff in health care environments). This service robot, referred to as the Adaptive Robotic Nurse Assistant (ARNA), was developed to enhance the productivity of nurses through cooperation during physical tasks (eg, patient walking, item fetching, object delivery) as well as nonphysical tasks (eg, patient observation and feedback). This pilot study evaluated the acceptability of ARNA to provide ambulatory assistance to patients. METHODS: We conducted a trial with 24 participants to collect data and address the following research question: Is the use of ARNA as an ambulatory assistive device for patients acceptable to nurses? The experiments were conducted in a simulated hospital environment. Nursing students (as proxies for nursing staff) were grouped in dyads, with one participant serving as a nurse and the other acting as a patient. Two questionnaires were developed and administrated to the participants based on the Technology Acceptance Model with respect to the two subscales of perceived usefulness and perceived ease of use metrics. In order to evaluate the internal consistency/reliability of the questionnaires, we calculated Cronbach alpha coefficients. Furthermore, statistical analyses were conducted to evaluate the relation of each variable in the questionnaires with the overall perceived usefulness and perceived ease of use metrics. RESULTS: Both Cronbach alpha values were acceptably high (.93 and .82 for perceived usefulness and perceived ease of use questionnaires, respectively), indicating high internal consistency of the questionnaires. The correlation between the variables and the overall perceived usefulness and perceived ease of use metrics was moderate. The average perceived usefulness and perceived ease of use metrics among the participants were 4.13 and 5.42, respectively, out of possible score of 7, indicating a higher-than-average acceptability of this service robot. CONCLUSIONS: The results served to identify factors that could affect nurses' acceptance of ARNA and aspects needing improvement (eg, flexibility, ease of operation, and autonomy level).

Central Retinal Artery Occlusion Following Prone Transcranial Surgery for Craniosynostosis and Discussion of Risk Factors.

Vision loss is a known rare complication of prone positioning during surgery. Vision loss following prone surgery is most commonly attributed to direct pressure on the eye but can also be caused by central retinal artery occlusion (CRAO) in the absence of pressure on the eye. Central retinal artery occlusion has not been previously described following prone transcranial surgery for craniosynostosis. We present two cases of monocular CRAO following prone calvarial expansion. A multidisciplinary root cause analysis suggested that raised intracranial pressure and intraoperative tranexamic acid may have been risk factors for the development of CRAO in these cases as no conventional risk factors for CRAO following prone surgery were present. Because of this, we retrospectively reviewed all prone transcranial procedures performed at the Oxford Craniofacial Unit for the presence of raised intracranial pressure and intraoperative tranexamic acid use. A total of 662 prone procedures have been performed between 1994 and March, 2019. Tranexamic acid has been used routinely in all transcranial procedures since 2012 and in the last 311 consecutive prone cases. Fifty-one (7.7%) prone procedures were performed for raised intracranial pressure, and tranexamic acid was used in the 33 most recent of these. Since the implementation of standard intraoperative administration of tranexamic acid there have been 2 cases of CRAO following prone surgery. The overall incidence of CRAO was 0.3% but was 6% in the context of raised intracranial pressure and tranexamic acid use. Prone positioning raised intracranial pressure and tranexamic acid use together may represent a potent combination of risk factors for CRAO.

COVID-19-Hospitalized Patients in Karnataka: Survival and Stay Characteristics.

The information on the clinical course of coronavirus disease 2019 (COVID-19) and its correlates which are essential to assess the hospital care needs of the population are currently limited. We investigated the factors associated with hospital stay and death for COVID-19 patients for the entire state of Karnataka, India. A retrospective-cohort analysis was conducted on 445 COVID-19 patients that were reported in the publicly available media-bulletin from March 9, 2020, to April 23, 2020, for the Karnataka state. This fixed cohort was followed till 14 days (May 8, 2020) for definitive outcomes (death/discharge). The median length of hospital stay was 17 days (interquartile range: 15-20) for COVID-19 patients. Having severe disease at the time of admission (adjusted-hazard-ratio: 9.3 (3.2-27.3);P < 0.001) and being aged ≥ 60 years (adjusted-hazard-ratio: 11.9 (3.5-40.6);P < 0.001) were the significant predictors of COVID-19 mortality. By moving beyond descriptive (which provide only crude information) to survival analyses, information on the local hospital-related characteristics will be crucial to model bed-occupancy demands for contingency planning during COVID-19 pandemic.

p53 gain-of-function mutations increase Cdc7-dependent replication initiation.

Cancer-associated p53 missense mutants confer gain of function (GOF) and promote tumorigenesis by regulating crucial signaling pathways. However, the role of GOF mutant p53 in regulating DNA replication, a commonly altered pathway in cancer, is less explored. Here, we show that enhanced Cdc7-dependent replication initiation enables mutant p53 to confer oncogenic phenotypes. We demonstrate that mutant p53 cooperates with the oncogenic transcription factor Myb in vivo and transactivates Cdc7 in cancer cells. Moreover, mutant p53 cells exhibit enhanced levels of Dbf4, promoting the activity of Cdc7/Dbf4 complex. Chromatin enrichment of replication initiation factors and subsequent increase in origin firing confirm increased Cdc7-dependent replication initiation in mutant p53 cells. Further, knockdown of CDC7 significantly abrogates mutant p53-driven cancer phenotypes in vitro and in vivo Importantly, high CDC7 expression significantly correlates with p53 mutational status and predicts poor clinical outcome in lung adenocarcinoma patients. Collectively, this study highlights a novel functional interaction between mutant p53 and the DNA replication pathway in cancer cells. We propose that increased Cdc7-dependent replication initiation is a hallmark of p53 gain-of-function mutations.

A case of central nervous system vasculitis presenting as a mass-like lesion.

Vasculitis of the central nervous system presenting as a mass lesion is a relatively uncommon occurrence. Even more uncommon is a vasculitis mimicking a demyelinating lesion. We present here an interesting case of a 15-year-old boy who was found to have a mass-like lesion on neuroimaging involving the left subcortical white matter and deep gray matter. The differential diagnosis for this lesion was primary demyelination versus a glial tumor, the former being more favored over the latter. Biopsy of this lesion however revealed findings compatible with a vasculitis, which was unexpected given the neuroimaging findings. To the authors' knowledge, case reports in the English literature of a vasculitic lesion mimicking demyelination are scarce. This case also serves as a reminder of the diagnostic difficulty that arises in a pediatric patient with an initial presentation of mass-like lesion.

Receptor tyrosine kinases (RTKs) in breast cancer: signaling, therapeutic implications and challenges.

Breast cancer is a multifactorial disease and driven by aberrant regulation of cell signaling pathways due to the acquisition of genetic and epigenetic changes. An array of growth factors and their receptors is involved in cancer development and metastasis. Receptor Tyrosine Kinases (RTKs) constitute a class of receptors that play important role in cancer progression. RTKs are cell surface receptors with specialized structural and biological features which respond to environmental cues by initiating appropriate signaling cascades in tumor cells. RTKs are known to regulate various downstream signaling pathways such as MAPK, PI3K/Akt and JAK/STAT. These pathways have a pivotal role in the regulation of cancer stemness, angiogenesis and metastasis. These pathways are also imperative for a reciprocal interaction of tumor and stromal cells. Multi-faceted role of RTKs renders them amenable to therapy in breast cancer. However, structural mutations, gene amplification and alternate pathway activation pose challenges to anti-RTK therapy.