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SARS-CoV-2, a novel human coronavirus, has created a global disease burden infecting > 100 million humans in just over a year. RT-PCR is currently the predominant method of diagnosing this viral infection although a variety of tests to detect viral antigens have also been developed. In this study, we adopted a SISCAPA-based enrichment approach using anti-peptide antibodies generated against peptides from the nucleocapsid protein of SARS-CoV-2. We developed a targeted workflow in which nasopharyngeal swab samples were digested followed by enrichment of viral peptides using the anti-peptide antibodies and targeted parallel reaction monitoring (PRM) analysis using a high-resolution mass spectrometer. This workflow was applied to 41 RT-PCR-confirmed clinical SARS-CoV-2 positive nasopharyngeal swab samples and 30 negative samples. The workflow employed was highly specific as none of the target peptides were detected in negative samples. Further, the detected peptides showed a positive correlation with the viral loads as measured by RT-PCR Ct values. The SISCAPA-based platform described in the current study can serve as an alternative method for SARS-CoV-2 viral detection and can also be applied for detecting other microbial pathogens directly from clinical samples. Supplementary Information The online version contains supplementary material available at 10.1186/s12014-021-09331-z.

Background NFPTs are the most common operated pituitary tumours and can present with visual field defects, hormone deficiencies and headache. Surgery is treatment of choice but recurrence rate is high ranging from 10-50%, depending on the extent of tumour removal. No confirmed predictive biomarkers for NFPT recurrence have been identified, apart from Ki-67. We applied high-throughput mass spectrometry-based phosphoproteomic approach to explore the phosphorylation pattern of proteins in NFPTs in order to identify predictive markers of invasion and recurrence. Methods Based on radiological, histopathological, and surgical features, NFPTs were sub-grouped into three groups: non-invasive (n=5), invasive (n=10) and recurrent (n=5) subtypes. Invasiveness was determined by radiology (Knosp classification 3&4), histopathological invasion (bone, dura and mucosa) and intraoperative findings. Tumour recurrence was based on radiological data for a mean±SD follow-up of112±39 months. Fresh-frozen pituitary tumour tissues were used for protein extraction and phosphopeptides were enriched using TiO 2 and labelled with tandem mass tags and subjected tomass spectrometry (Orbitrap)for quantification. Candidate hyper-phosphorylated proteins were validated by immunohistochemistry in 200 additional tumour samples by immunoblotting (n=36). Results In total, we identified 3185 phosphopeptides and observed significant difference in phosphorylation levels of invasive and recurrent groups. Compared to non-invasive cases, in invasive group we found, 452 hyper and 93 hypo phosphorylated proteins, while in the recurrent group there were 790 hyper and 307 hypo phosphorylated proteins. Phospho-serine showed the highest level of difference (90.3%) among the groups, followed by threonine (8.9%) and tyrosine (0.8%). One of the top differentially phosphorylated proteins was Ser552 of β-catenin showing significant hyper-phosphorylation in recurrent (p Conclusions our study has identified hyper-phosphorylation of β-catenin at the Ser552 in recurrent and invasive NFPT subgroups. The H-score of β-catenin p552 correlates with tumour recurrence free survival in a large cohort of NFPT patients, which supports that β-catenin pSer552 could be used as predictive biomarker for NFPT recurrence. Presentation: No date and time listed

Overexpression and amplification of AXL receptor tyrosine kinase (RTK) has been found in several hematologic and solid malignancies. Activation of AXL can enhance tumor-promoting processes such as cancer cell proliferation, migration, invasion and survival. Despite the important role of AXL in cancer development, a deep and quantitative mapping of its temporal dynamic signaling transduction has not yet been reported. Here, we used a TMT labeling-based quantitative proteomics approach to characterize the temporal dynamics of the phosphotyrosine proteome induced by AXL activation. We identified &gt, 1100 phosphotyrosine sites and observed a widespread upregulation of tyrosine phosphorylation induced by GAS6 stimulation. We also detected several tyrosine sites whose phosphorylation levels were reduced upon AXL activation. Gene set enrichment-based pathway analysis indicated the activation of several cancer-promoting and cell migration/invasion-related signaling pathways, including RAS, EGFR, focal adhesion, VEGFR and cytoskeletal rearrangement pathways. We also observed a rapid induction of phosphorylation of protein tyrosine phosphatases, including PTPN11 and PTPRA, upon GAS6 stimulation. The novel molecules downstream of AXL identified in this study along with the detailed global quantitative map elucidating the temporal dynamics of AXL activation should not only help understand the oncogenic role of AXL, but also aid in developing therapeutic options to effectively target AXL.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the worldwide COVID-19 pandemic which began in 2019. It has a high transmission rate and pathogenicity leading to health emergencies and economic crisis. Recent studies pertaining to the understanding of the molecular pathogenesis of SARS-CoV-2 infection exhibited the indispensable role of ion channels in viral infection inside the host. Moreover, machine learning (ML)-based algorithms are providing higher accuracy for host-SARS-CoV-2 protein-protein interactions (PPIs). In this study, predictions of PPIs of SARS-CoV-2 proteins with human ion channels (HICs) were performed using PPI-MetaGO algorithm. The PPIs were predicted with 82.71% accuracy, 84.09% precision, 84.09% sensitivity, 0.89 AUC-ROC, 65.17% Matthews correlation coefficient (MCC) score and 84.09% F1 score. Thereafter, PPI networks of SARSCoV-2 proteins with HICs were generated. Furthermore, biological pathway analysis of HICs interacting with SARS-CoV-2 proteins showed the involvement of six pathways, namely inflammatory mediator regulation of transient receptor potential (TRP) channels, insulin secretion, renin secretion, gap junction, taste transduction and apelin signaling pathway. Our analysis suggests that transient receptor potential cation channel subfamily M member 4 (TRPM4), transient receptor potential cation channel subfamily A member 1 (TRPA1), gap junction protein alpha 1 (GJA1), potassium calcium-activated channel subfamily N member 4 (KCNN4), acid sensing ion channel subunit 1 (ASIC1) and inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) could serve as an initial set to the experimentalists for further validation. Additionally, various US food and drug administration (FDA) approved drugs interacting with the potential HICs were also identified. The study also reinforcesthe drug repurposing approach for the development of host directed antiviral drugs.

Since the recent outbreak of COVID-19, there have been intense efforts to understand viral pathogenesis and host immune response to combat SARS-CoV-2. It has become evident that different host alterations can be identified in SARS-CoV-2 infection based on whether infected cells, animal models or clinical samples are studied. Although nasopharyngeal swabs are routinely collected for SARS-CoV-2 detection by RT-PCR testing, host alterations in the nasopharynx at the proteomic level have not been systematically investigated. Thus, we sought to characterize the host response through global proteome profiling of nasopharyngeal swab specimens. A mass spectrometer combining trapped ion mobility spectrometry (TIMS) and high-resolution QTOF mass spectrometer with parallel accumulation-serial fragmentation (PASEF) was deployed for unbiased proteome profiling. First, deep proteome profiling of pooled nasopharyngeal swab samples was performed in the PASEF enabled DDA mode, which identified 7723 proteins that were then used to generate a spectral library. This approach provided peptide level evidence of five missing proteins for which MS/MS spectrum and mobilograms were validated with synthetic peptides. Subsequently, quantitative proteomic profiling was carried out for 90 individual nasopharyngeal swab samples (45 positive and 45 negative) in DIA combined with PASEF, termed as diaPASEF mode, which resulted in a total of 5023 protein identifications. Of these, 577 proteins were found to be upregulated in SARS-CoV-2 positive samples. Functional analysis of these upregulated proteins revealed alterations in several biological processes including innate immune response, viral protein assembly, and exocytosis. To the best of our knowledge, this study is the first to deploy diaPASEF for quantitative proteomic profiling of clinical samples and shows the feasibility of adopting such an approach to understand mechanisms and pathways altered in diseases.

Non-receptor tyrosine kinases represent an important class of signaling molecules which are involved in driving diverse cellular pathways. Although, the large majority have been well-studied in terms of their protein-binding partners, the interactomes of some of the key non-receptor tyrosine kinases such as TNK2 (also known as activated Cdc42-associated kinase 1 or ACK1) have not been systematically investigated. Aberrant expression and hyperphosphorylation of TNK2 has been implicated in a number of cancers. However, the exact proteins and cellular events that mediate phenotypic changes downstream of TNK2 are unclear. Biological systems that employ proximity-dependent biotinylation methods, such as BioID, are being increasingly used to map protein-protein interactions as they provide increased sensitivity in discovering interaction partners. In this study, we employed BioID coupled to the biotinylation site identification technology (BioSITe) method that we recently developed to perform molecular mapping of intracellular proteins associated with TNK2. We also employed stable isotope labeling with amino acids in cell culture (SILAC) to quantitatively explore the interactome of TNK2. By performing a controlled comparative analysis between full-length TNK2 and its truncated counterpart, we were not only able to confidently identify site-level biotinylation of previously well-established TNK2 binders and substrates such as NCK1, NCK2, CTTN, STAT3, but also discover several novel TNK2 interacting partners. We validated TNK2 interaction with one of the novel TNK2 interacting protein, clathrin interactor 1 (CLINT1), using immunoblot analysis. Overall, this work reveals the power of the BioSITe method coupled to BioID and highlights several molecules that warrant further exploration to assess their functional significance in TNK2-mediated signaling.

Background The COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in a short time. Methods Here, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigen from nasopharyngeal swab samples. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric waveform ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assay on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was developed using fragment ion intensities from the PRM data. Findings The optimized targeted assay, which was used to analyze 88 positive and 88 negative nasopharyngeal swab samples for validation, resulted in 98% (95% CI = 0.922–0.997) (86/88) sensitivity and 100% (95% CI = 0.958–1.000) (88/88) specificity using RT-PCR-based molecular testing as the reference method. Interpretation Our results demonstrate that direct detection of infectious agents from clinical samples by tandem mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories, which has hitherto been limited to analysis of pure microbial cultures. Funding This study was supported by DBT/Wellcome Trust India Alliance Margdarshi Fellowship grant IA/M/15/1/502023 awarded to AP and the generosity of Eric and Wendy Schmidt.

We study the ensembles of direct product of $m$ random unitary matrices of size $N$ drawn from a given circular ensemble. We calculate the statistical measures, viz. number variance and spacing distribution to investigate the level correlations and fluctuation properties of the eigenangle spectrum. Similar to the random unitary matrices, the level statistics is stationary for the ensemble constructed by their direct product. We find that the eigenangles are uncorrelated in the small spectral intervals. While, in large spectral intervals, the spectrum is rigid due to strong long-range correlations between the eigenangles. The analytical and numerical results are in good agreement. We also test our findings on the multipartite system of quantum kicked rotors.

Alzheimer’s disease (AD) is the most common cause of dementia and is associated with serious neurologic sequelae resulting from neurodegenerative changes. Identification of markers of early-stage AD could be important for designing strategies to arrest the progression of disease. The brain is rich in lipids because they are crucial for signal transduction and anchoring of membrane proteins. Cerebrospinal fluid (CSF) is an excellent specimen for studying the metabolism of lipids in AD because it can reflect changes occurring in the brain. We aimed to identify CSF lipidomic alterations associated with AD, using untargeted lipidomics, carried out in positive and negative ion modes. We found CSF lipids that were significantly altered in AD cases. In addition, comparison of CSF lipid profiles between persons with mild cognitive impairment (MCI) and AD showed a strong positive correlation between the lipidomes of the MCI and AD groups. The novel lipid biomarkers identified in this study are excellent candidates for validation in a larger set of patient samples and as predictive biomarkers of AD through future longitudinal studies. Once validated, the lipid biomarkers could lead to early detection, disease monitoring and the ability to measure the efficacy of potential therapeutic interventions in AD. Cerebrospinal fluid lipid biomarkers of Alzheimer’s disease were discovered using tandem mass spectrometry.

Background We evaluated the analytical sensitivity and specificity of 4 rapid antigen diagnostic tests (Ag RDTs) for severe acute respiratory syndrome coronavirus 2, using reverse transcription quantitative PCR (RT-qPCR) as the reference method and further characterizing samples using droplet digital quantitative PCR (ddPCR) and a mass spectrometric antigen test. Methods Three hundred fifty (150 negative and 200 RT-qPCR positive) residual PBS samples were tested for antigen using the BD Veritor lateral flow (LF), ACON LF, ACON fluorescence immunoassay (FIA), and LumiraDx FIA. ddPCR was performed on RT-qPCR–positive samples to quantitate the viral load in copies/mL applied to each Ag RDT. Mass spectrometric antigen testing was performed on PBS samples to obtain a set of RT-qPCR–positive, antigen-positive samples for further analysis. Results All Ag RDTs had nearly 100% specificity compared to RT-qPCR. Overall analytical sensitivity varied from 66.5% to 88.3%. All methods detected antigen in samples with viral load >1 500 000 copies/mL RNA, and detected ≥75% of samples with viral load of 500 000 to 1 500 000 copies/mL. The BD Veritor LF detected only 25% of samples with viral load between 50 000 to 500 000 copies/mL, compared to 75% for the ACON LF device and >80% for LumiraDx and ACON FIA. The ACON FIA detected significantly more samples with viral load Conclusions Ag RDTs differ significantly in analytical sensitivity, particularly at viral load

Parkinson's disease (PD) is the second most common neurodegenerative disorder that results in motor dysfunction and, eventually, cognitive impairment. α-Synuclein protein is known as a central protein to the pathophysiology of PD, but the underlying pathological mechanism still remains to be elucidated. In an effort to understand how α-synuclein underlies the pathology of PD, various PD mouse models with α-synuclein overexpression have been developed. However, systemic analysis of the brain proteome of those mouse models is lacking. In this study, we established two mouse models of PD by injecting α-synuclein preformed fibrils (PFF) or by inducing overexpression of human A53T α-synuclein to investigate common pathways in the two different types of the PD mouse models. For more accurate quantification of mouse brain proteome, the proteins were quantified using the method of stable isotope labeling with amino acids in mammals . We identified a total of 8355 proteins from the two mouse models; ∼6800 and ∼7200 proteins from α-synuclein PFF-injected mice and human A53T α-synuclein transgenic mice, respectively. Through pathway analysis of the differentially expressed proteins common to both PD mouse models, it was discovered that the complement and coagulation cascade pathways were enriched in the PD mice compared to control animals. Notably, a validation study demonstrated that complement component 3 (C3)-positive astrocytes were increased in the ventral midbrain of the intrastriatal α-synuclein PFF-injected mice and C3 secreted from astrocytes could induce the degeneration of dopaminergic neurons. This is the first study that highlights the significance of the complement and coagulation pathways in the pathogenesis of PD through proteome analyses with two sophisticated mouse models of PD.

Keratoconus (KCN) is a leading cause for cornea grafting worldwide. Keratoconus is a multifactorial disease that causes progressive thinning of the cornea and whose etiology is poorly understood. Several studies have used proteomics on patient tear fluids to identify potential biomarkers. However, proteome of the cornea itself has not been investigated fully. We report here new findings from a case–control study using multiplexed mass spectrometry (MS) on individual (unpooled) corneas to gain deeper insights into proteins and biomarkers relevant to keratoconus. We employed a high-pressure approach to extract total protein from individual corneas from five cases and five controls, followed by trypsin digestion and tandem mass tag (TMT) labeling. The MS-derived data were searched using the Human NCBI RefSeq protein database v92, with peptides and proteins filtered at 1% false discovery rate. A total of 3132 proteins were detected, of which 627 were altered significantly (p ≤ 0.05) in keratoconus corneas. The increases were overwhelmingly in the mTOR/PI3/AKT signal-mediated regulations of cell survival and proliferation, nonsense-mediated decay of transcripts, and proteasomal pathways. The decreases were in several extracellular matrix proteins and in many members of the complement system. Importantly, this multiplexed proteomic study of keratoconus corneas identified, to our knowledge, the largest number of corneal proteins. The novel findings include changes in pathways that regulate transcript stability, proteasomal degradation, and the complement system in corneas with keratoconus. These observations offer new prospects toward future discovery of novel molecular targets for diagnostic and therapeutic innovations for patients with keratoconus.

Cancer-associated mutations in the spliceosome gene SF3B1 create a neomorphic protein that produces aberrant mRNA splicing in hundreds of genes, but the ensuing biologic and therapeutic consequences of this missplicing are not well understood. Here we have provided evidence that aberrant splicing by mutant SF3B1 altered the transcriptome, proteome, and metabolome of human cells, leading to missplicing-associated downregulation of metabolic genes, decreased mitochondrial respiration, and suppression of the serine synthesis pathway. We also found that mutant SF3B1 induces vulnerability to deprivation of the nonessential amino acid serine, which was mediated by missplicing-associated downregulation of the serine synthesis pathway enzyme PHGDH. This vulnerability was manifest both in vitro and in vivo, as dietary restriction of serine and glycine in mice was able to inhibit the growth of SF3B1(MUT) xenografts. These findings describe a role for SF3B1 mutations in altered energy metabolism, and they offer a new therapeutic strategy against SF3B1(MUT) cancers.

AlN nucleation layers (NL) with different thickness were grown on 4H-SiC substrates using MOVPE. The growth evolution of the AlN layer on SiC was investigated. The effect of fully strained AlN nucleation layer (NL) on the AlN/SiC interface and on the subsequent GaN growth was also investigated. The thickness of the NL was varied between 10 nm and 100 nm in order to study the surface morphology and strain of AlN NL layer. The interface quality between AlN and SiC was characterized using HRXRD. It was observed that 100 nm NL was partially relaxed whereas 40 nm AlN NL was fully strained with improved SiC/AlN interface. GaN layer grown with fully strained NL showed improved surface morphology and lower screw dislocation density. 2DEG properties of AlGaN/GaN HEMT structures grown on fully strained and partially relaxed NL were found to be almost similar.

The pre-erythrocytic liver stage of the malaria parasite, comprising sporozoites and the liver stages into which they develop, remains one of the least understood parts of the lifecycle, in part owing to the low numbers of parasites. Nonetheless, it is recognized as an important target for antimalarial drugs and vaccines. Here we provide the first proteomic analysis of merosomes, which define the final phase of the liver stage and are responsible for initiating the blood stage of infection. We identify a total of 1879 parasite proteins, and a core set of 1188 proteins quantitatively detected in every biological replicate, providing an extensive picture of the protein repertoire of this stage. This unique data set will allow us to explore key questions about the biology of merosomes and hepatic merozoites.

Proteomics research today no longer simply seeks exhaustive protein identification; increasingly, it is also desirable to obtain robust, large-scale quantitative information. To accomplish this, data-independent acquisition (DIA) has emerged as a promising strategy largely owing to developments in advanced mass spectrometers and sophisticated data analysis methods. Nevertheless, the highly complex multiplexed MS/MS spectra produced by DIA remain challenging to interpret. Here, we present a novel strategy to analyze DIA data, based on unambiguous precursor mass assignment through the mPE-MMR (multiplexed post-experimental monoisotopic mass refinement) procedure and combined with complementary multistage database searching. Compared to conventional spectral library searching, the accuracy and sensitivity of peptide identification were significantly increased by incorporating precise precursor masses in DIA data. We demonstrate identification of additional peptides absent from spectral libraries, including sample-specific mutated peptides and post-translationally modified peptides using MS-GF+ and MODa/MODi multistage database searching. This first use of unambiguously determined precursor masses to mine DIA data demonstrates considerable potential for further exploitation of this type of experimental data.

Primary immunodeficiencies (PIDs) are a rare and heterogeneous group of inherited genetic disorders that are characterized by an absent or impaired immune system. In this report, we describe the use of next-generation sequencing to investigate a male infant with clinical and immunological manifestations suggestive of a PID. Whole-exome sequencing of the infant along with his parents revealed a novel nucleotide variant (cytosine to adenine substitution at nucleotide position 252) in the coding region of the interleukin 2 receptor subunit gamma (IL2RG) gene. The mother was found to be a carrier. These findings are consistent with a diagnosis of X-linked severe combined immunodeficiency and represent the first such reported mutation in an Indian family. This mutation leads to an asparagine to lysine substitution (p.Asn84Lys) located in the extracellular domain of IL2RG, which is predicted to be pathogenic. Our study demonstrates the power of next-generation sequencing in identifying potential causative mutations to enable accurate clinical diagnosis, prenatal screening, and carrier female detection in PID patients. We believe that this approach, which is not a current routine in clinical practice, will become a mainstream component of individualized medicine in the near future.

Here, we demonstrates the fabrication of binder free and very efficient supercapacitor electrode based on tungsten nitride (W2N) thin film on stainless steel (SS) substrate using reactive sputtering technique. W2N thin film as a working electrode exhibits high specific capacitance (163 F g−1 at 0.5 mA cm−2 in 1 M H2SO4) along with excellent cycling stability. The binder free symmetric supercapacitor (W2N||W2N) device delivers a high specific capacitance (80 Fg-1) and long life span (90.46% capacitance retention after 10,000 cycles) along with high energy (12.92 Whkg−1) and power (∼674 kWkg−1 at 9.36 Whkg−1) densities. These observed excellent electrochemical performances of the present W2N thin film based supercapacitor device, recommend it as a potential candidate for energy storage applications.

The presence of residual stress is inevitable and major constraint for MEMS devices as they induce deformation, fracture, fatigue and micro structural changes in the structure. This paper presents the influence of residual stresses (up to − 2100 MPa) on the performance of AlN based piezoelectric MEMS accelerometer structure. The MEMS structure consists of an AlN coated silicon proof mass (800 µm × 800 µm × 300 µm) suspended by four beams (400 µm × 200 µm × 10 µm). Under the influence of residual stress (− 2100 MPa), the first three modal frequencies (13.54 kHz, 20.95 kHz and 20.95 kHz) of the accelerometer structure are reduced to 5.19 kHz, 10.17 kHz and 10.17 kHz respectively. The location of proof mass deflection and maximum von-Mises stress also get altered. Hence the structure sensitivity is also modified from 3.64 × 10−2/g (at no residual stress) to 2.54 × 10−4/g (at − 2100 MPa). The damping factor of the structure also gets modified from 3.96 to 10.38 due to the residual stress. Thus, the accelerometer frequency response (3 dB bandwidth) is found to be diminished from 1.81 to 0.26 kHz due to the presence of residual stress.

Nanostructured CuO thin films were synthesized by thermal evaporation and annealing. Structural, optical and morphological changes in the CuO film upon annealing and their overall impact on its photocatalytic activity were investigated employing X-ray diffraction, UV–Vis absorption spectroscopy, atomic force microscopy and field emission scanning electron microscopy. Significant modifications in the morphological, optical, structural and photocatalytic behavior of nanostructured CuO thin film were observed upon thermal annealing. Thermal annealing led to the growth of CuO nanoparticles and the average size of CuO nanoparticles increased from 23 nm to 293 nm as the annealing temperature was increased to 600 o C. CuO thin film sample annealed at 400 °C exhibited superior photocatalytic activities over other samples for the degradation of malachite green and methylene blue dyes in 120 and 160 min, respectively. The improved photocatalytic behavior of CuO thin film annealed at 400 °C is attributed to its narrower band gap, improved utilization of sunlight and enhanced adsorption of dye due to increased surface area arising from formation of CuO nanoparticles and their aggregates at the surface.

Gallbladder cancer (GBC) is a rare malignancy, associated with poor disease prognosis with a 5-year survival of only 20%. This has been attributed to late presentation of the disease, lack of early diagnostic markers and limited efficacy of therapeutic interventions. Elucidation of molecular events in GBC can contribute to better management of the disease by aiding in the identification of therapeutic targets. To identify aberrantly activated signaling events in GBC, tandem mass tag-based quantitative phosphoproteomic analysis of five GBC cell lines was carried out. Proline-rich Akt substrate 40 kDa (PRAS40) was one of the proteins found to be hyperphosphorylated in all the invasive GBC cell lines. Tissue microarray-based immunohistochemical labeling of phospho-PRAS40 (T246) revealed moderate to strong staining in 77% of the primary gallbladder adenocarcinoma cases. Regulation of PRAS40 activity by inhibiting its upstream kinase PIM1 resulted in a significant decrease in cell proliferation, colony forming and invasive ability of GBC cells. Our results support the role of PRAS40 phosphorylation in GBC cell survival and aggressiveness. This study also elucidates phospho-PRAS40 as a clinical marker in GBC and the role of PIM1 as a therapeutic target in GBC. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12079-018-00503-5) contains supplementary material, which is available to authorized users.

The aluminum nitride (AlN) powder/thin films were prepared from an aluminum–urea complex. The complex, hexa urea aluminate(III) chloride, has proven to be a potential single source precursor to aluminum nitrides because urea molecules construct a coordination sphere around the metal atom and form a stable structure, compared with air sensitive halide and hydride precursors. The precursor and the spin coated thin films of the precursor on quartz and Si(100) substrates were pyrolysed at various temperatures (800 °C to 1000 °C) and pressure (100 Torr to 1 Torr) under nitrogen atmosphere. The pyrolysed powders/films were characterized by FTIR (Fourier Transform Infrared) spectroscopy, TGA (Thermogravimetric analysis), PXRD (Powder X-ray Diffraction), FESEM (Field emission scanning electron microscopy), UV-Vis (Ultra violet-Visible spectroscopy), etc. The XRD results show that the polycrystalline aluminum nitride was obtained at a temperature of 1000 °C and at 1 Torr pressure. The average crystallites size calculated from XRD were in the range of 20–10 nm, which decreased with increase of pyrolysis temperature. We also conducted the pyrolysis process under argon instead of nitrogen and found that most of the nitrogen came from urea for the formation of AlN but the nitrogen atmosphere favoured clean formation of AlN. The band gaps of the deposited films were found in the range of 5.1–6.2 eV.

PIK3CA is one of the most frequently mutated genes in human cancers, with the two most prevalent activating mutations being E545K and H1047R. Although the altered intracellular signaling pathways in these cells have been described, the effect of these mutations on their extracellular vesicles (EVs) has not yet been reported. To study altered cellular physiology and intercellular communication through proteomic analysis of EVs, MCF10A cells and their isogenic mutant versions (PIK3CA E545K and H1047R) were cultured and their EVs enriched by differential ultracentrifugation. Proteins were extracted, digested with trypsin and the peptides labeled with tandem mass tag (TMT) reagents and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Four thousand six hundred and fifty-five peptides were identified from 579 proteins of which 522 proteins have been previously described in EVs. Relative quantitation revealed altered levels of EV proteins including several cell adhesion molecules. Mesothelin, E-cadherin, and epithelial cell adhesion molecule were elevated in both mutant cell-derived EVs. Markers of tumor invasion and progression like galectin-3 and transforming growth factor beta induced protein were increased in both mutants. Overall, activating mutations in PIK3CA result in altered EV composition with characteristic changes associated with these hotspot mutations.

Background: Cholangiocarcinoma is an aggressive malignancy with limited systemic therapeutics. We have recently identified LCK, a non-receptor tyrosine kinase, as a potential therapeutic target in select subtypes of cholangiocarcinoma. Proteomic profiling in other cancer types has proven beneficial in predicting response to systemic therapies. Herein, we present proteomic profiling of cholangiocarcinoma can predict sensitivity to a novel LCK inhibitor in patient derived xenograft models. Methods: Proteins from 28 unique cholangiocarcinoma patient derived xenograft models (PDX) were isolated, labeled with tandem mass tags (TMT) and then analyzed by liquid chromatography and mass spectroscopy (LC/MS). Phosphopeptides were separately evaluated both by Immobilized Metal Affinity Chromatography and pY-enrichment techniques. Following normalization, the global proteome, serine/threonine phosphoproteome, and tyrosine phosphoproteome were analyzed and a proteomic signature was created for each tumor. These signatures were compared to PDX models with known response profiles to NTRC0652-0, an LCK inhibitor (1 resistant and 1 sensitive). The top 200 differentially abundant proteins (log2FC 2.6) were then utilized to develop a sensitive and a resistant signature. Correlation indices were then calculated across the 28 PDX model library. An additional predicted resistant and predicted sensitive model was expanded. Tumor bearing mice were randomized based on tumor volume to either treatment with NTRC (30mg/kg PO daily), n=5, or vehicle, n=4, (10% Kolliphor, 10% DMSO, 80% Water, 2 mol equivalents HCl). Tumors were measured weekly with calipers and then weighed at the end of the study. Results: In the top 200 differentially abundant proteins that represented the proteomic signature there was over representation of proteins with decreased abundance. There were only 12 proteins with higher abundance in the sensitive model as compared to the resistant. Utilizing the signature developed from the global proteomics data, nine tumors were predicted to be sensitive while 17 were predicted to be resistant to NTRC. PDX115 is a an intrahepatic CCA PDX known to contain an IDH1 mutation and was predicted to be resistant by modeling. The model was resistant to single agent NTRC with no demonstrable effect on tumor growth in vivo. PDX175 is a distal CCA PDX and was predicted to be sensitive by modeling. In vivo growth was inhibited by NTRC as compared to vehicle treated mice following two weeks of treatment (983% vs 435%, p=0.03). Additionally, final tumor weight was significantly lower in NTRC treated mice (0.651 g vs 0.317 g, p=0.04). Conclusion: Cholangiocarcinoma proteomic signatures can be used to predict therapeutic response to a novel LCK inhibitor in preclinical models. Whether or not this could be applied to other treatment modalities in cholangiocarcinoma remains to be determined. Citation Format: Ryan Watkins, Roberto Alva-Ruiz, Caitlin Conboy, Dong-Gi Mun, Erik Jessen, Diep Vu, Jos de Man, Rogier Buijsman, Akhilesh Pandey, Mitesh Borad, Gregory Gores, Rory Smoot. Proteomic profiling of cholangiocarcinoma predicts response to a novel small molecule inhibitor in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3094.

Background: As a key receptor tyrosine kinase (RTK) regulating tumorigenesis, AXL is overexpressed in multiple different types of cancers. AXL can be activated by homodimerization after binding with its ligand, growth arrest specific 6 protein (Gas6) or by heterodimerization with other RTKs. AXL overexpression and/or activation have been reported to promote cancer cell proliferation, migration/invasion and to induce resistance to targeted therapy. However, the AXL downstream signaling network has not yet been fully elucidated. Methods: We employed a CRISPR-CAS9-based knockout technology to create AXL inducible knockout cells in a human triple negative breast cancer (TNBC) cell line, HCC1395. In order to explore the downstream signaling events driven by AXL, we induced AXL knockout for 72 hours and used a 6-plex TMT-labeling-based quantitative proteomic approach to characterize the protein phosphorylation alterations induced by AXL knockout. IMAC and anti-phosphotyrosine antibody (pY1000)-based phosphopeptide enrichment methods were used to comprehensively quantify the phosphoproteome changes induced by AXL knockout. Results: In this study, we identified 38,525 phospho serine/threonine sites and 1,427 phosphotyrosine sites. In response to AXL knockout, 1,581 phospho serine/threonine sites and 40 phosphotyrosine sites showed significant differences between AXL-KO and AXL-WT groups with >1.5-fold change and p Conclusion: Quantitative analysis of the phosphoproteome driven by AXL demonstrated that AXL plays a pivotal role in regulating canonical oncogenic signaling but also signaling pathways involved in RNA processing and DNA repair. Our data also suggest that targeting AXL could suppress these oncogenic signaling pathways and have therapeutic potential to improve clinical outcomes for patients with AXL overexpression. Citation Format: Li Wang, Nicole Pearson, Yuning Xiong, Santosh Renuse, Ran Cheng, Jodi M. Carter, Akhilesh Pandey, Xinyan Wu. Quantitative phosphoproteomic analysis of AXL signaling network in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3913.

The pituitary function is regulated by a complex system involving the hypothalamus and biological networks within the pituitary. Although the hormones secreted from the pituitary have been well studied, comprehensive analyses of the pituitary proteome are limited. Pituitary proteomics is a field of postgenomic research that is crucial to understand human health and pituitary diseases. In this context, we report here a systematic proteomic profiling of human anterior pituitary gland (adenohypophysis) using high-resolution Fourier transform mass spectrometry. A total of 2164 proteins were identified in this study, of which 105 proteins were identified for the first time compared with high-throughput proteomic-based studies from human pituitary glands. In addition, we identified 480 proteins with secretory potential and 187 N-terminally acetylated proteins. These are the first region-specific data that could serve as a vital resource for further investigations on the physiological role of the human anterior pituitary glands and the proteins secreted by them. We anticipate that the identification of previously unknown proteins in the present study will accelerate biomedical research to decipher their role in functioning of the human anterior pituitary gland and associated human diseases.

Even though SYK and ZAP70 kinases share high sequence homology and serve analogous functions, their expression in B and T cells is strictly segregated throughout evolution. Here, we identified aberrant ZAP70 expression as a common feature in a broad range of B cell malignancies. We validated SYK as the kinase that sets the thresholds for negative selection of autoreactive and premalignant clones. When aberrantly expressed in B cells, ZAP70 competes with SYK at the BCR signalosome and redirects SYK from negative selection to tonic PI3K signaling, thereby promoting B cell survival. In genetic mouse models for B-ALL and B-CLL, conditional expression of Zap70 accelerated disease onset, while genetic deletion impaired malignant transformation. Inducible activation of Zap70 during B cell development compromised negative selection of autoreactive B cells, resulting in pervasive autoantibody production. Strict segregation of the two kinases is critical for normal B cell selection and represents a central safeguard against the development of autoimmune disease and B cell malignancies. acceptedVersion

Peptides presented by MHC molecules on the cell surface, or the immunopeptidome, play an important role in the adaptive arm of the immune response. Antigen processing for MHC class I molecules is a ubiquitous pathway present in all nucleated cells which generates and presents peptides of both self and non-self-origin. Peptides with post-translational modifications represent one category of peptides presented by MHC class I molecules. However, owing to the complexity of self-peptides presented by cells, the diversity of peptides with post-translational modifications is not well-studied. In this study, we carried out MHC Class I immunopeptidomics analysis of Loucy T-cell leukemia and A375 malignant melanoma cell line to characterize the diversity of post-translational modifications of MHC class I-bound peptides. Using high resolution mass spectrometry, we identified 25,761 MHC-bound peptides across both cell lines using Bolt and Sequest search engines. The enrichment method was highly specific as ~ 90% of the peptides were of typical length (8-12 amino acids long) and the motifs were expected based on previously reported motifs for MHC I alleles. Among the MHC-bound peptides, we identified phosphorylation as a major post-translational modification followed by deamidation. We observed site-specific localization of these post-translational modifications, at position P4 for phosphorylated peptides and position P3 for deamidated peptides. We identified a smaller number of peptides with acetylated and methylated lysine, possibly due to very low stoichiometric levels of these PTMs compared to phosphorylation and deamidation. Using PEAKS de novo sequencing algorithm, we identified spliced peptides that accounted for ~ 5-7% of MHC-bound peptides that were otherwise similar in their features as normal MHC-bound peptides. We validated the identity of several post-translationally modified peptides and spliced peptides through mass spectrometric analysis of synthetic peptides. Our study confirms post-translationally modified peptides to be present at low stoichiometric levels along with unusual spliced peptides through unbiased identification using high resolution mass spectrometry.The online version contains supplementary material available at 10.1007/s42485-021-00066-x.

Shukla-Vernon syndrome (SHUVER) is an extremely rare neurodevelopmental disorder characterized by global developmental delay, intellectual disability, behavioral anomalies, and dysmorphic features. Pathogenic variants in the BCORL1 gene have been identified as the molecular cause for this disorder. The BCORL1 gene encodes for BCL-6 corepressor-like protein 1, a transcriptional corepressor that is an integral component of protein complexes involved in transcription repression. In this study, we report an Indian family with two male siblings with features of Shukla-Vernon syndrome. The patients exhibited global developmental delay, intellectual disability, kyphosis, seizures, and dysmorphic features including bushy prominent eyebrows with synophrys, sharp beaked prominent nose, protuberant lower jaw, squint, and hypoplastic ears with fused ear lobes. No behavioral abnormalities were observed. Whole exome sequencing revealed a novel potentially pathogenic arginine to cysteine substitution (p.Arg1265Cys) in the BCORL1 protein. This is the second report of Shukla-Vernon syndrome with a novel missense variant in the BCORL1 gene. Our study confirms and expands the phenotypes and genotypes described previously for this syndrome and should aid in diagnosis and genetic counselling of patients and their families.

Acute Kidney Injury (AKI) after percutaneous nephrolithotomy (PNL) is a significant complication, but evidence on its incidence is bereft in the literature. The objective of this prospective observational study was to analyze the incidence of post-PNL AKI and the potential risk factors and outcomes. Demographic data collected included age, gender, body mass index (BMI), comorbidities (hypertension, diabetes mellitus), and drug history—particularly angiotensin converting enzyme inhibitors (ACE inhibitors), angiotensin II receptor blockers and beta blockers. Laboratory data included serial serum creatinine measured pre- and postoperation (12, 24, and 48 h), hemoglobin (Hb), total leucocyte count (TLC), Prothrombin time (PT), serum uric acid and urine culture. Stone factors were assessed by noncontrast computerized tomography of kidneys, ureter and bladder (NCCT KUB) and included stone burden, location and Hounsfield values. Intraoperative factors assessed were puncture site, tract size, tract number, operative time, the need for blood transfusion and stone clearance. Postoperative complications were documented using the modified Clavien–Dindo grading system and patients with postoperative AKI were followed up with serial creatinine measurements up to 1 year. Among the 509 patients analyzed, 47 (9.23%) developed postoperative AKI. Older patients, with associated hypertension and diabetes mellitus, those receiving ACE inhibitors and with lower preoperative hemoglobin and higher serum uric acid, had higher incidence of AKI. Higher stone volume and density, staghorn stones, multiple punctures and longer operative time were significantly associated with postoperative AKI. Patients with AKI had an increased length of hospital stay and 17% patients progressed to chronic kidney disease (CKD). Cut-off values for patient age (39.5 years), serum uric acid (4.05 mg/dL) and stone volume (673.06 mm3) were assessed by receiver operating characteristic (ROC) curve analysis. Highlighting the strong predictors of post-PNL AKI allows early identification, proper counseling and postoperative planning and management in an attempt to avoid further insult to the kidney.

SUMMARY Mitochondrial carriers (MCs) mediate the passage of small molecules across the inner mitochondrial membrane (IMM), enabling regulated crosstalk between compartmentalized reactions. Despite MCs representing the largest family of solute carriers in mammals, most have not been subjected to a comprehensive investigation, limiting our understanding of their metabolic contributions. Here, we functionally characterize SFXN1, a member of the non-canonical, sideroflexin family. We find that SFXN1, an integral IMM protein with an uneven number of transmembrane domains, is a TIM22 complex substrate. SFXN1 deficiency leads to mitochondrial respiratory chain impairments, most detrimental to complex III (CIII) biogenesis, activity, and assembly, compromising coenzyme Q levels. The CIII dysfunction is independent of one-carbon metabolism, the known primary role for SFXN1 as a mitochondrial serine transporter. Instead, SFXN1 supports CIII function by participating in heme and α-ketoglutarate metabolism. Our findings highlight the multiple ways that SFXN1-based amino acid transport impacts mitochondrial and cellular metabolic efficiency., Graphical Abstract, In brief Acoba et al. show that the amino acid transporter SFXN1 is a cargo of the TIM22 translocon that is important for maintaining complex III function and supports coenzyme Q, heme, and α-ketoglutarate metabolism.

The dynamic modification of specific serine and threonine residues of intracellular proteins by O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) mitigates injury and promotes cytoprotection in a variety of stress models. The O-GlcNAc transferase (OGT) and the O-GlcNAcase are the sole enzymes that add and remove O-GlcNAc, respectively, from thousands of substrates. It remains unclear how just two enzymes can be specifically controlled to affect glycosylation of target proteins and signaling pathways both basally and in response to stress. Several lines of evidence suggest that protein interactors regulate these responses by affecting OGT and O-GlcNAcase activity, localization, and substrate specificity. To provide insight into the mechanisms by which OGT function is controlled, we have used quantitative proteomics to define OGT’s basal and stress-induced interactomes. OGT and its interaction partners were immunoprecipitated from OGT WT, null, and hydrogen peroxide–treated cell lysates that had been isotopically labeled with light, medium, and heavy lysine and arginine (stable isotopic labeling of amino acids in cell culture). In total, more than 130 proteins were found to interact with OGT, many of which change their association upon hydrogen peroxide stress. These proteins include the major OGT cleavage and glycosylation substrate, host cell factor 1, which demonstrated a time-dependent dissociation after stress. To validate less well-characterized interactors, such as glyceraldehyde 3-phosphate dehydrogenase and histone deacetylase 1, we turned to parallel reaction monitoring, which recapitulated our discovery-based stable isotopic labeling of amino acids in cell culture approach. Although the majority of proteins identified are novel OGT interactors, 64% of them are previously characterized glycosylation targets that contain varied domain architecture and function. Together these data demonstrate that OGT interacts with unique and specific interactors in a stress-responsive manner., Graphical Abstract, Highlights • O-GlcNAc levels change dynamically in response to injury. • Injury does not induce changes in activity of the enzymes that cycle O-GlcNAc. • Quantitative proteomics identified and validated interactors of OGT. • The interactome of OGT changes significantly in response to oxidative stress., In Brief The goal of these studies was to provide insight into the regulation of the O-GlcNAc transferase (OGT) basally and in response to oxidative stress, as well as the role that O-GlcNAc plays in promoting cytoprotection. Using quantitative proteomics, the basal and injury-induced interactome of OGT has been defined and validated. Protein interactors are anticipated to regulate either the activity or substrate targeting of OGT, or to be substrates of OGT, thus affecting cytoprotection.

The nuclear lamina provides structure to the nucleus and serves as an interface between the cytoskeleton and large heterochromatin domains called LADs. This study describes the microproteome of this LAD/lamina interface., The nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, the so-called lamina-associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions. In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2β to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin-directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify both LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina, identified putative LAD proximal proteins and found several proteins that appear to interface with both micro-proteomes. Importantly, several proteins essential for LAD function, including heterochromatin regulating proteins related to H3K9 methylation, were identified in this study.

BACKGROUND Sodium-glucose co-transporter (SGLT) inhibitors reduce heart failure (HF) hospitalization and cardiovascular death in several populations, including patients with type 2 diabetes mellitus, renal impairment, HF and cardiovascular disease. The efficacy of SGLT inhibitors in patients with atrial fibrillation/flutter (AF) and their effect on incidence or recurrence of AF has yet to be systematically assessed. Our objectives were to determine whether sodium-glucose co-transporter (SGLT) inhibitors reduce atrial fibrillation/flutter (AF) and whether a history of AF modifies the effect of SGLT inhibitors on the composite of heart failure hospitalization or cardiovascular death. METHODS AND RESULTS We searched MEDLINE, Embase and CENTRAL to March 2021. Pairs of reviewers identified randomized controlled trials that compared an SGLT inhibitor to placebo or no therapy. The primary outcome was the rate of AF reported as an adverse event. The secondary outcome was a composite of hospitalization/urgent visit for HF or cardiovascular death, stratified by history of AF at baseline. We assessed risk of bias using the Cochrane tool and the overall quality of evidence using GRADE. Thirty eligible trials reported on AF events (71,553 participants, mean age 62 years, 35.6% women). Moderate quality evidence supported a lower risk of AF events with SGLT inhibitors (1.0% versus 1.4%; risk ratio 0.78; 95% confidence interval [CI] 0.68-0.89; I2=0%). Three trials reported on the secondary composite outcome (18,826 participants, mean age 66 years, 38.1% women). In patients with a history of AF, SGLT inhibitors resulted in a lower risk in the composite of HF hospitalization or cardiovascular death (hazard ratio [HR] 0.70; 95% CI 0.57-0.85; I2=0%) – similar to the effect estimate for patients without AF (HR 0.70; 95% CI 0.61-0.79; I2=0%), p-value for interaction: 1.00. CONCLUSION SGLT inhibitors may reduce the incidence or recurrence of AF. These drugs reduce a composite of HF hospitalization or cardiovascular death, both in patients with and without AF.

DKK3 is a secreted protein, which belongs to a family of Wnt antagonists and acts as a potential tumor suppressor in gallbladder cancer. To further understand its tumor suppressor functions, we overexpressed DKK3 in 3 GBC cell lines. We have employed high-resolution mass spectrometry and tandem mass tag (TMT) multiplexing technology along with immobilized metal affinity chromatography to enrich phosphopeptides to check the downstream regulators. In this study, we reported for the first time the alteration in the phosphorylation of 14 kinases upon DKK3 overexpression. In addition, we observed DKK3 induced hyper phosphorylation of 2 phosphatases: PPP1R12A and PTPRA, which have not been reported previously. Canonical pathway analysis of altered molecules indicated differential enrichment of signaling cascades upon DKK3 overexpression in all the 3 cell lines. Protein kinase A signaling, Sirtuin signaling pathway, and Cell Cycle Control of Chromosomal Replication were observed to be differentially activated in the GBC cell lines. Our study revealed, DKK3 overexpression has differential effect based on the aggressive behavior of the cell lines. This study expands the understanding of DKK3-mediated signaling events and can be used as a primary factor for understanding the complex nature of this molecule.

Aluminum nitride-based MEMS resonators are one of the interesting recent research topics for its tremendous potential in a wide variety of applications. This paper focuses on the detrimental effect of residual stress on the AlN based MEMS resonator design for acoustic applications. The residual stress in the sputtered c axis () preferred oriented AlN layers on Si (111) substrates are studied as a function of layer thickness. The films exhibited compressive residual stresses at different thickness values: -1050 MPa (700 nm), -500 MPa (900 nm), and -230 MPa (1200 nm). A mushroom-shaped AlN based piezoelectric MEMS resonator structure has been designed for the different AlN layer thicknesses. The effect of the residual stresses on the mode shapes, resonant frequencies, and quality factor (Q) of the resonator structures are studied. The resonant frequency of the structures are altered from 235 kHz, 280 kHz, and 344 kHz to 65 kHz, 75 kHz and 371 kHz due to the residual stress of -1050 MPa (thickness: 700 nm), -500 MPa (thickness: 900 nm) and -230 MPa (thickness: 1200 nm) respectively. At no residual stress, the quality factors of the resonator structures are 248, 227, 241 corresponding to the 700 nm, 900 nm, and 1200 nm thick AlN layers respectively. The presence of the residual stress reduced the Q values from 248 (thickness: 700 nm), 227 (thickness: 900 nm), 241 (thickness: 1200 nm) to 28, 53, and 261 respectively.

We have investigated the impact of AlN buffer layer growth parameters for developing highly single crystalline AlGaN films. The low mobility of Al adatoms and high temperature for compound formation are amongst the major causes that affects the growth quality of AlGaN films. Thus, proper optimization need to be carried out for achieving high quality AlGaN due to an augmented tendency of defect generation compared to GaN films. Thus, growth conditions need to be amended to maximize the incorporation ability of adatoms and minimize defect density. So, this study elaborates the growth optimization of AlGaN/AlN/Si (111) heterostructure with varied AlN buffer growth temperature (760 to 800 °C). It was observed that the remnant Al in low temperature growth of AlN buffer layer resist the growth quality of AlGaN epitaxial films. A highly single crystalline AlGaN film with comparatively lowest rocking curve FWHM value (~ 0.61°) and smooth surface morphology with least surface defect states was witnessed when AlN buffer was grown at 780 °C. From the Vegard’s law, the photoluminescence analysis unveils Aluminium composition of 31.5% with significantly reduced defect band/NBE band ratio to 0.3. The study demonstrates good crystalline quality AlGaN film growth with Aluminium content variation between ~ 30–39% in AlGaN/AlN heterostructure on Si(111) substrate leading to a bandgap range which is suitable for next-generation solar-blind photodetection applications.

Mammalian red blood cells (RBCs), which primarily contain hemoglobin, exemplify an elaborate maturation process, with the terminal steps of RBC generation involving extensive cellular remodeling. This encompasses alterations of cellular content through distinct stages of erythroblast maturation that result in the expulsion of the nucleus (enucleation) followed by the loss of mitochondria and all other organelles and a transition to anaerobic glycolysis. Whether there is any link between erythroid removal of the nucleus and the function of any other organelle, including mitochondria, remains unknown. Here we demonstrate that mitochondria are key to nuclear clearance. Using live and confocal microscopy and high-throughput single-cell imaging, we show that before nuclear polarization, mitochondria progressively move toward one side of maturing erythroblasts and aggregate near the nucleus as it extrudes from the cell, a prerequisite for enucleation to proceed. Although we found active mitochondrial respiration is required for nuclear expulsion, levels of mitochondrial activity identify distinct functional subpopulations, because terminally maturing erythroblasts with low relative to high mitochondrial membrane potential are at a later stage of maturation, contain greatly condensed nuclei with reduced open chromatin–associated acetylation histone marks, and exhibit higher enucleation rates. Lastly, to our surprise, we found that late-stage erythroblasts sustain mitochondrial metabolism and subsequent enucleation, primarily through pyruvate but independent of in situ glycolysis. These findings demonstrate the critical but unanticipated functions of mitochondria during the erythroblast enucleation process. They are also relevant to the in vitro production of RBCs as well as to disorders of the erythroid lineage.

Pathogenic alterations in the DPM2 gene have been previously described in patients with hypotonia, progressive muscle weakness, absent psychomotor development, intractable seizures, and early death. We identified biallelic DPM2 variants in a 23-year-old male with truncal hypotonia, hypertonicity, congenital heart defects, intellectual disability, and generalized muscle wasting. His clinical presentation was much less severe than that of the three previously described patients. This is the second report on this ultra-rare disorder. Here we review the characteristics of previously reported individuals with a defect in the DPM complex while expanding the clinical phenotype of DPM2-Congenital Disorders of Glycosylation. In addition, we offer further insights into the pathomechanism of DPM2-CDG disorder by introducing glycomics and lipidomics analysis. ispartof: MOLECULAR GENETICS AND METABOLISM vol:132 issue:1 pages:27-37 ispartof: location:United States status: published

Nanoparticles (NPs) are particulate substances having 1 to 100 nm size. Owing to their extremely small size, NPs exhibit typical structural and biological characteristics, which include a larger reactive surface area and the ability to pass through cell and tissue barriers making them useful for humans in medicine, agriculture, and industrial applications. In addition to beneficial effects, gradually increasing applications and exposure of NPs might lead to harmful effects because of oxidative stress, inflammation, DNA damage, and eventually causing cell death by necrosis or apoptosis. This chapter highlights the interaction of nanomaterials with cellular system and molecular mechanisms involved in NP-mediated adverse effects as well as strategies to overcome NP-mediated toxicity.

Convolutional neural networks (CNNs) are workhorses of deep learning. A popular architecture in CNN is Residual Net (ResNet) that emphasizes on learning a residual mapping rather than directly fit input to output. Subsequent to ResNet, Squeeze and Excitation Network (SENet) introduced a squeeze and excitation block (SE block) on every residual mapping of ResNet to improve its performance. The SE block quantifies the importance of each feature map and weights them accordingly. In this work, we propose a new architecture SEANet built over SENet by introducing an aggregate block after SE block. We choose sum as the aggregate operation. The aggregation helps in minimizing redundancies in feature representation and provide a global feature representation across feature maps by downsampling their number. We demonstrate the superior performance of our SEANet over ResNet and SENet on benchmark CIFAR-10 and CIFAR-100 datasets. Specifically, SEANet reduces classification error rate on CIFAR-10 by around 2% and 3%, respectively, over ResNet and SENet. On CIFAR-100, SEANet reduces error by around 5% and 9% when compared against ResNet and SENet. Further, SEANet outperforms the latest EncapNet and both its variants EncapNet+ and EncapNet++ on CIFAR-100 dataset by around 2%.

Glycans decorate almost all proteins and lipids on the cell surface, and they play a critical role in cell-cell interactions. Terminal sialic acid addition to glycans helps the immune system distinguish between “self” and “non-self.” The enzyme ST8sia6 adds alpha-2,8 disialic acids to cell surface receptors, and this sialic acid addition promotes binding to the inhibitory receptor Siglec-E. We have previously shown ST8sia6 overexpression in tumors accelerates tumor growth in a Siglec-E dependent manner. Proteomic analysis in murine tumor lines identified CD44 as a target for ST8sia6. CD44 expression on tumors is associated with metastasis and chemoresistance. Since humans and mice have different sialic acid structures, we examined whether ST8sia6 makes CD44 a ligand to the ortholog of Siglec-E in human cells, Siglec-7. When HEK293T cells overexpress ST8sia6 and CD44 together, Siglec-7 binding was significantly increased compared to ST8sia6 or CD44 overexpression alone. Thus, ST8sia6 has a conserved role from mouse to human in decorating CD44 with sialic acid. Our study proposes that ST8sia6 sialylation of CD44 could suppress immune responses and enables cancer progression in human. Supported by grant: 2T32AI007425-23 to DJF and R01 CA243545-01S1 to V.S.S.