bluuebunny/crossref_metadata_2025 huggingface dataset:
Eliminating fibroblast activation protein-α expressing cells by photoimmunotheranostics
Authors: Jin Jiefu , Barnett James D. , Krishnamachary Balaji , Mironchik Yelena , Luo Catherine K. , Kobayashi Hisataka , Bhujwalla Zaver M.
Published: November 2021

Photoimmunotherapy (PIT) using an antibody conjugated to a near infrared dye IR700 is achieving significant success in target-specific elimination of cells. Fibroblast activation protein alpha (FAP-α) is an important target in cancer because of its expression by cancer associated fibroblasts (CAFs) as well as by some cancer cells. CAFs that express FAP-α have protumorigenic and immune suppressive functions. Using immunohistochemistry of human breast cancer tissue microarrays, we identified an increase of FAP-α+ CAFs in invasive breast cancer tissue compared to adjacent normal tissue. We found FAP-α expression increased in fibroblasts co-cultured with cancer cells. In proof-of-principle studies, we engineered human FAP-α overexpressing MDA-MB-231 and HT-1080 cancer cells and murine FAP-α overexpressing NIH-3T3 fibroblasts to evaluate several anti-FAP-α antibodies and selected AF3715 based on its high binding-affinity with both human and mouse FAP-α. After conjugation of AF3715 with the phthalocyanine dye IR700, the resultant antibody conjugate, FAP-α-IR700, was evaluated in cells and tumors for its specificity and effectiveness in eliminating FAP-α expressing cell populations with PIT. FAP-α-IR700-PIT resulted in effective FAP-α-specific cell killing in the engineered cancer cells and in two patient-derived CAFs in a dose-dependent manner. Following an intravenous injection, FAP-α-IR700 retention was three-fold higher than IgG-IR700 in FAP-α overexpressing tumors, and two-fold higher compared to wild-type tumors. FAP-α-IR700-PIT resulted in significant growth inhibition of tumors derived from FAP-α overexpressing human cancer cells. A reduction of endogenous FAP-α+ murine CAFs was identified at 7 days after FAP-α-IR700-PIT. FAP-α-targeted NIR-PIT presents a promising strategy to eliminate FAP-α+ CAFs.
Weighted Shoes in the Wild: Initial Insights into the Relationship Between the Effort of Walking and the Amount of Walking Performed
Authors: Wu Mailing R. , Adamczyk Peter G. , Collins Steven H.
Published: November 2021

Purpose

Walking comprises a large portion of active energy expenditure in humans. Interventions that increase or decrease the energy expended on each step, such as ankle weights or energy-saving orthoses, may therefore strongly impact fitness. The overall effect of increasing or decreasing per-step energy use is unclear, however, because people may choose to walk less or more, respectively, in response.

Methods

In this study, healthy college students with normal body mass index wore weighted and unweighted shoes embedded with an inertial measurement unit for one week each. Community-based walking data were analyzed for number of steps, distance traveled and walking speed. Oxygen consumption using each set of shoes at a range of speeds were measured in a laboratory setting and used to estimate metabolic energy expended during community-based walking. A survey measured subjective response to each pair of shoes.

Results

The weighted shoes increased per-step energy cost by about 26%. Subjects strongly disliked the weighted shoes (P = 0.001) and found them tiring (P = 0.003). Despite this dislike, subjects did not significantly reduce distance walked (P = 0.6), number of steps (P = 0.7), or average speed (P = 0.9) compared to normal shoes. This led to a small but not statistically significant increase in energy expended during walking over a five-day period (12.3 ± 9.6% increase, P = 0.2). On the final collection day this trend appeared to reverse, with fewer steps taken and lower metabolic energy expended with the weighted shoes. Twenty-four subjects were recruited but only ten completed the protocol, with dislike of the weighted shoe condition being the primary reason for dropout.

Conclusions

Increasing the energy cost of each step led to greater energy expended through walking. However, there are indications that behavioral changes would be greater with a longer intervention or increased retention. For example, the large dropout rate suggests that some subjects avoided walking with the weighted shoes entirely, simply by leaving the study. Follow-on studies among patient groups may reveal a fitness benefit to either increasing or reducing the energy cost of walking.
Induction of virus-induced gene silencing andin plantavalidation in cucurbits using the CFMMV-Cm vector
Authors: Rhee Sun-Ju , Jang Yoon Jeong , Park Jun-Young , Lee Gung Pyo
Published: November 2021

Virus-induced gene silencing (VIGS) has been employed for the high-throughput analysis of endogenous gene function. We developed a CaMV 35S promoter-driven cucumber fruit mottle mosaic virus-Cm vector (pCF93) for the efficient generation of viral transcripts in plants. Using the novel pCF93 vector, we identified genes related to male sterility in watermelon (Citrullus lanatus), which is recalcitrant to genetic transformation. We previously reported reference-based andde novotranscriptomic profiling for the detection of differentially expressed genes between a male fertile line (DAH3615) and its near isogenic male sterile line (DAH3615-MS). Based on the RNA-seq results, we identified 38de novo-exclusive differentially expressed genes (DEDEGs) that are potentially responsible for male sterility. Partial genes of 200∼300bp were cloned into pCF93 which was then inoculated into DAH, a small type of watermelon that enables high-throughput screening with a small cultivation area. In this manner, we simultaneously characterized phenotypes associated with the 38 candidate genes in a common-sized greenhouse. Eight out of the 38 gene-silenced plants produced male sterile flowers with abnormal stamens and no pollens. Gene expression levels in flowers were validated via RT-qPCR. Stamen histological sections from male sterile floral buds and mature flowers showed developmental disruption and shrunken pollen sacs. Based on the current findings, we believe that the novel pCF93 vector and our VIGS system facilitate high-throughput analysis for the study of gene function in watermelons.

One sentence summary

The CaMV 35S promoter-driven cucumber fruit mottle mosaic virus-Cm vector (pCF93) facilitates large-scale validation of male sterility-related gene functions in watermelon.
Allele segregation analysis of F1 hybrids between independent Brassica allohexaploid lineages
Authors: Quezada-Martinez Daniela , Zou Jun , Zhang Wenshan , Meng Jinling , Batley Jacqueline , Mason Annaliese S.
Published: November 2021

In the Brassica genus we find both diploid species (one genome) and allotetraploid species (two different genomes) but no naturally occurring hexaploid species (three different genomes, AABBCC). Although hexaploids can be produced via human intervention, these neo- polyploids have quite unstable genomes and usually suffer from severe genome reshuffling. Whether these genome rearrangements continue in later generations and whether genomic arrangements follow similar, reproducible patterns between different lines is still unknown. We crossed Brassica hexaploids resulting from different species combinations to produce five F1 hybrids, and analyzed the karyotypes of the parents and the F1 hybrids, as well as allele segregation in a resulting test-cross population via molecular karyotyping using SNP array genotyping. Although some genomic regions were found to be more likely to be duplicated, deleted or rearranged, a consensus pattern was not shared between genotypes. Brassica hexaploids had a high tolerance for fixed structural rearrangements, but which rearrangements occur and become fixed over many generations does not seem to show either strong reproducibility or to indicate selection for stability. On average, we observed 10 de novo chromosome rearrangements contributed almost equally from both parents to the F1 hybrids. At the same time, the F1 hybrid meiosis produced on average 8.6 new rearrangements. Hence, the increased heterozygosity in the F1 hybrid did not significantly improve genome stability in our hexaploid hybrids, and might have had the opposite effect. However, hybridization between lineages was readily achieved and may be exploited for future genetics and breeding purposes.
Cell Painting predicts impact of lung cancer variants
Authors: Caicedo Juan C. , Arevalo John , Piccioni Federica , Bray Mark-Anthony , Hartland Cathy L. , Wu Xiaoyun , Brooks Angela N. , Berger Alice H. , Boehm Jesse S. , Carpenter Anne E. , Singh Shantanu
Published: November 2021

Most variants in most genes across most organisms have an unknown impact on the function of the corresponding gene. This gap in knowledge is especially acute in cancer, where clinical sequencing of tumors now routinely reveals patient-specific variants whose functional impact on the corresponding gene is unknown, impeding clinical utility. Transcriptional profiling was able to systematically distinguish these variants of unknown significance (VUS) as impactful vs. neutral in an approach called expression-based variant-impact phenotyping (eVIP). We profiled a set of lung adenocarcinoma-associated somatic variants using Cell Painting, a morphological profiling assay that captures features of cells based on microscopy using six stains of cell and organelle components. Using deep-learning-extracted features from each cell’s image, we found that cell morphological profiling (cmVIP) can predict variants’ functional impact and, particularly at the single-cell level, reveals biological insights into variants which can be explored in our public online portal. Given its low cost, convenient implementation, and single-cell resolution, cmVIP profiling therefore seems promising as an avenue for using non-gene-specific assays to systematically assess the impact of variants, including disease-associated alleles, on gene function.
evSeq: Cost-Effective Amplicon Sequencing of Every Variant in a Protein Library
Authors: Wittmann Bruce J. , Johnston Kadina E. , Almhjell Patrick J. , Arnold Frances H.
Published: November 2021

Widespread availability of protein sequence-fitness data would revolutionize both our biochemical understanding of proteins and our ability to engineer them. Unfortunately, even though thousands of protein variants are generated and evaluated for fitness during a typical protein engineering campaign, most are never sequenced, leaving a wealth of potential sequence-fitness information untapped. This largely stems from the fact that sequencing is unnecessary for many protein engineering strategies; the added cost and effort of sequencing is thus unjustified. Here, we present every variant sequencing (evSeq), an efficient protocol for sequencing a variable region within every variant gene produced during a protein engineering campaign at a cost of cents per variant. Execution of evSeq is simple, requires no sequencing experience to perform, relies only on resources and services typically available to biology labs, and slots neatly into existing protein engineering workflows. Analysis of evSeq data is likewise made simple by its accompanying software (found atgithub.com/fhalab/evSeq, documentation atfhalab.github.io/evSeq), which can be run on a personal laptop and was designed to be accessible to users with no computational experience. Low-cost and easy to use, evSeq makes collection of extensive protein variant sequence-fitness data practical.
TCR-BERT: learning the grammar of T-cell receptors for flexible antigen-xbinding analyses
Authors: Wu Kevin , Yost Kathryn E. , Daniel Bence , Belk Julia A. , Xia Yu , Egawa Takeshi , Satpathy Ansuman , Chang Howard Y. , Zou James
Published: November 2021

The T-cell receptor (TCR) allows T-cells to recognize and respond to antigens presented by infected and diseased cells. However, due to TCRs’ staggering diversity and the complex binding dynamics underlying TCR antigen recognition, it is challenging to predict which antigens a given TCR may bind to. Here, we present TCR-BERT, a deep learning model that applies self-supervised transfer learning to this problem. TCR-BERT leverages unlabeled TCR sequences to learn a general, versatile representation of TCR sequences, enabling numerous downstream applications. We demonstrate that TCR-BERT can be used to build state-of-the-art TCR-antigen binding predictors with improved generalizability compared to prior methods. TCR-BERT simultaneously facilitates clustering sequences likely to share antigen specificities. It also facilitates computational approaches to challenging, unsolved problems such as designing novel TCR sequences with engineered binding affinities. Importantly, TCR-BERT enables all these advances by focusing on residues with known biological significance. TCR-BERT can be a useful tool for T-cell scientists, enabling greater understanding and more diverse applications, and provides a conceptual framework for leveraging unlabeled data to improve machine learning on biological sequences.
Algorithmic Parameter Estimation and Uncertainty Quantification for Hodgkin-Huxley Neuron Models
Authors: Wang Y. Curtis , Sinha Nirvik , Rudi Johann , Velasco James , Idumah Gideon , Powers Randall K. , Heckman Charles J. , Chardon Matthieu
Published: November 2021

Experimental data-based parameter search for Hodgkin–Huxley-style (HH) neuron models is a major challenge for neuroscientists and neuroengineers. Current search strategies are often computationally expensive, are slow to converge, have difficulty handling nonlinearities or multimodalities in the objective function, or require good initial parameter guesses. Most important, many existing approaches lack quantification of uncertainties in parameter estimates even though such uncertainties are of immense biological significance. We propose a novel method for parameter inference and uncertainty quantification in a Bayesian framework using the Markov chain Monte Carlo (MCMC) approach. This approach incorporates prior knowledge about model parameters (as probability distributions) and aims to map the prior to a posterior distribution of parameters informed by both the model and the data. Furthermore, using the adaptive parallel tempering strategy for MCMC, we tackle the highly nonlinear, noisy, and multimodal loss function, which depends on the HH neuron model. We tested the robustness of our approach using the voltage trace data generated from a 9-parameter HH model using five levels of injected currents (0.0, 0.1, 0.2, 0.3, and 0.4 nA). Each test consisted of running the ground truth with its respective currents to estimate the model parameters. To simulate the condition for fitting a frequency-current (F-I) curve, we also introduced an aggregate objective that runs MCMC against all five levels simultaneously. We found that MCMC was able to produce many solutions with acceptable loss values (e.g., for 0.0 nA, 889 solutions were within 0.5% of the best solution and 1,595 solutions within 1% of the best solution). Thus, an adaptive parallel tempering MCMC search provides a “landscape” of the possible parameter sets with acceptable loss values in a tractable manner. Our approach is able to obtain an intelligently sampled global view of the solution distributions within a search range in a single computation. Additionally, the advantage of uncertainty quantification allows for exploration of further solution spaces, which can serve to better inform future experiments.
Nonquantal Transmission at the Vestibular Hair Cell-Calyx Synapse: KLV Currents Modulate Fast Electrical and Slow K+ Potentials in the Synaptic Cleft
Authors: Govindaraju Aravind Chenrayan , Quraishi Imran H. , Lysakowski Anna , Eatock Ruth Anne , Raphael Robert M.
Published: November 2021

Vestibular hair cells transmit information about head position and motion across synapses to primary afferent neurons. At some of these synapses, the afferent neuron envelopes the hair cell, forming an enlarged synaptic terminal called a calyx. The vestibular hair cell-calyx synapse supports a mysterious form of electrical transmission that does not involve gap junctions termed nonquantal transmission (NQT). The NQT mechanism is thought to involve the flow of ions from the pre-synaptic hair cell to the post-synaptic calyx through low-voltage-activated channels driven by changes in cleft [K+] as K+ exits the hair cell. However, this hypothesis has not been tested with a quantitative model and the possible role of an electrical potential in the cleft has remained speculative. Here we present a computational model that captures salient experimental observations of NQT and identifies overlooked features that corroborate the existence of an electrical potential (ϕ) in the synaptic cleft. We show that changes in cleft ϕ reduce transmission latency and illustrate the relative contributions of both cleft [K+] and ϕ to the gain and phase of NQT. We further demonstrate that the magnitude and speed of NQT depend on calyx morphology and that increasing calyx height reduces action potential latency in the calyx afferent. These predictions are consistent with the idea that the calyx evolved to enhance NQT and speed up vestibular signals that drive neural circuits controlling gaze, balance, and orientation.

Significance Statement

The ability of the vestibular system to drive the fastest reflexes in the nervous system depends on rapid transmission of mechanosensory signals at vestibular hair cell synapses. In mammals and other amniotes, afferent neurons form unusually large calyx terminals on certain hair cells, and communication at these synapses includes nonquantal transmission (NQT), which avoids the synaptic delay of quantal transmission. We present a quantitative model that shows how NQT depends on the extent of the calyx covering the hair cell and attributes the short latency of NQT to changes in synaptic cleft electrical potential caused by current flowing through open potassium channels in the hair cell. This previously undescribed mechanism may act at other synapses.
Analytical kinetic model of native tandem promoters in E. coli
Authors: Chauhan Vatsala , Bahrudeen Mohamed N.M. , Palma Cristina S.D. , Baptista Ines S.C. , Almeida Bilena L.B. , Dash Suchintak , Kandavalli Vinodh , Ribeiro Andre S.
Published: November 2021

Closely spaced promoters in tandem formation are abundant in bacteria. We investigated the evolutionary conservation, biological functions, and the RNA and single-cell protein expression of genes regulated by tandem promoters in E. coli. We also studied the sequence (distance between transcription start sites ‘dTSS’, pause sequences, and distances from oriC) and potential influence of the input transcription factors of these promoters. From this, we propose an analytical model of gene expression based on measured expression dynamics, where RNAP-promoter occupancy times and dTSS are the key regulators of transcription interference due to TSS occlusion by RNAP at one of the promoters (when dTSS ≤ 35 bp) and RNAP occupancy of the downstream promoter (when dTSS > 35 bp). Occlusion and downstream promoter occupancy are modeled as linear functions of occupancy time, while the influence of dTSS is implemented by a continuous step function, fit to in vivo data on mean single-cell protein numbers of 30 natural genes controlled by tandem promoters. The best-fitting step is at 35 bp, matching the length of DNA occupied by RNAP in the open complex formation. This model accurately predicts the squared coefficient of variation and skewness of the natural single-cell protein numbers as a function of dTSS. Additional predictions suggest that promoters in tandem formation can cover a wide range of transcription dynamics within realistic intervals of parameter values. By accurately capturing the dynamics of these promoters, this model can be helpful to predict the dynamics of new promoters and contribute to the expansion of the repertoire of expression dynamics available to synthetic genetic constructs.

Author Summary

Tandem promoters are common in nature, but investigations on their dynamics have so far largely relied on synthetic constructs. Thus, their regulation and potentially unique dynamics remain unexplored. We first performed a comprehensive exploration of the conservation of genes regulated by these promoters in E. coli and the properties of their input transcription factors. We then measured protein and RNA levels expressed by 30 Escherichia coli tandem promoters, to establish an analytical model of the expression dynamics of genes controlled by such promoters. We show that start site occlusion and downstream RNAP occupancy can be realistically captured by a model with RNAP binding affinity, the time length of open complex formation, and the nucleotide distance between transcription start sites. This study contributes to a better understanding of the unique dynamics tandem promoters can bring to the dynamics of gene networks and will assist in their use in synthetic genetic circuits.
Progressive sub-MIC Exposure ofKlebsiella pneumoniae43816 to Cephalothin Induces the Evolution of beta-lactam Resistance without Acquisition of beta-lactamase Genes
Authors: Anderson Jasmine R. , Lam Nghi B. , Jackson Jazmyne L. , Dorenkott Sean M. , Ticer Taylor , Maldosevic Emir , Velez Amanda , Camden Megan R. , Ellis Terri N.
Published: November 2021

Bacterial exposure to antibiotic concentrations below the minimum inhibitory concentration (MIC) may result in a selection window allowing for the rapid evolution of resistance. These sub-MIC concentrations are commonly found in the greater environment. This study aimed to evaluate the adaptive genetic changes inKlebsiella pneumoniae43816 after prolonged but increasing sub-MIC levels of the common antibiotic cephalothin over a fourteen-day period. Over the course of the experiment, antibiotic concentrations increased from 0.5 μg/mL to 7.5 μg/mL. At the end of this extended exposure, the final adapted bacterial culture exhibited clinical resistance to both cephalothin and tetracycline, altered cellular and colony morphology, and a highly mucoid phenotype. Cephalothin resistance exceeded 125 μg/mL without the acquisition of beta-lactamase genes. Whole genome sequencing identified a series of genetic changes that could be mapped over the fourteen-day exposure period to the onset of antibiotic resistance. Specifically, mutations in therpoBsubunit of RNA Polymerase, thetetR/acrRregulator, and thewcaJsugar transferase each fix at specific timepoints in the exposure regimen where the MIC susceptibility dramatically increases. These mutations indicate that alterations in the secretion of colanic acid and attachment of colonic acid to LPS, may contribute to the resistant phenotype. These data demonstrate that very low, sub-MIC concentrations of antibiotics can have dramatic impacts on the bacterial evolution of resistance. Additionally, this study demonstrates that beta-lactam resistance can be achieved through sequential accumulation of specific mutations without the acquisition of a beta-lactamase gene.

Importance

Bacteria are constantly exposed to low levels of antibiotics in the environment. The impact of this low-level exposure on bacterial evolution is not well understood. In this work, we developed a model to exposeKlebsiella pneumoniaeto progressive, low doses of the antibiotic cephalothin. After a fourteen-day exposure regimen, our culture exhibited full clinical resistance to this antibiotic without the traditional acquisition of inactivating genes. This culture also exhibited resistance to tetracycline, had a highly mucoid appearance, and exhibited altered, elongated cellular morphology. Whole genome sequencing identified a collection of mutations to the bacterial genome that could be mapped to the emergence of the resistant phenotype. This study demonstrates that antibiotic resistance can be achieved in response to low level antibiotic exposure and without the traditional acquisition of resistance genes. Further, this study identifies new genes that may play a role in the evolution of antibiotic resistant bacteria.
PDFGRα+ Stromal Cells Promote Salivary Gland Proacinar Differentiation Through FGF2-dependent BMP7 Signaling
Authors: Moskwa Nicholas , Mahmood Ayma , Nelson Deirdre A. , Altrieth Amber L. , Forni Paolo , Larsen Melinda
Published: November 2021

3.0

Stromal cells can direct epithelial differentiation during organ development; however, these pathways remain poorly defined. FGF signaling is essential for submandibular salivary gland development, and FGF2 can regulate proacinar cell differentiation in organoids through autocrine signaling in stromal cells. We performed scRNA Seq and identified stromal cell subsets expressing Fgf2 and Fgf10 that also express Pdgfrα. When combined with epithelial cells in organoids, MACS-sorted PDGFRα+ cells sufficiently promoted proacinar differentiation. Gene expression analysis revealed FGF2 activates the gene Bmp7 in the stroma. BMP7 could replace stromalsignaling and stimulate epithelial acinar differentiation but not branching. However, in the absence of FGF2, pathway analysis revealed that the stromal cells differentiated into myofibroblasts. Myofibroblast differentiation was induced when we treated organoids with TGFβ1, which also prevented proacinar differentiation. Conversely, FGF2 reversed TGFβ’s effects. Dissecting pathways driving acinar differentiation will facilitate development of regenerative therapies.

2.0

Summary Statement

Embryonic salivary glands contain multiple stromal cell populations. FGF2 maintains the stromal Pdgfrα+ population in-vitro. The PDGFRα+ stromal cells drive early epithelial secretory cell differentiation using BMP7.
ARL3 Mediates BBSome Ciliary Turnover by Promoting Its Outward Diffusion through the Transition Zone
Authors: Liu Yan-Xia , Sun Wei-Yue , Xue Bin , Zhang Rui-Kai , Li Wen-Juan , Xie Xixian , Fan Zhen-Chuan
Published: November 2021

Ciliary receptors and their certain downstream signaling components undergo intraflagellar transport (IFT) as BBSome cargoes to maintain their ciliary dynamics for sensing and transducing extracellular stimuli inside the cell. Cargo laden BBSomes shed from retrograde IFT at the proximal ciliary region above the transition zone (TZ) followed by diffusing through the TZ for ciliary retrieval, while how the BBSome barrier passage is controlled remains elusive. Here, we show that the BBSome is a major effector of the Arf-like 3 (ARL3) GTPase inChlamydomonas. Under physiological condition, ARL3GDPbinds the membrane for diffusing into and residing in cilia. Following a nucleotide conversion, ARL3GTPdissociates with the ciliary membrane and binds and recruits the IFT-detached and cargo (phospholipase D, PLD)-laden BBSome at the proximal ciliary region to diffuse through the TZ and out of cilia. ARL3 deficiency impairs ciliary signaling, e.g. phototaxis ofChlamydomonascells, by disrupting BBSome ciliary retrieval, providing a mechanistic understanding behind BBSome ciliary turnover required for ciliary signaling.
Inactivation effect and damage of multi-irradiance by UVCLED on Acinetobacter baumannii
Authors: Li Mengmeng , Zhao Baiqin , Hanlei  , Paste Wangzhen
Published: November 2021

It is acknowledged that the inactivation of ultraviolet has been widely used in various fields. Much literature has been reported that ultraviolet C caused DNA damage to achieve inactivation of microorganisms. There is a lack of unified dose calibration and related parameters in this field. In this study, we used a device consisted of the LED of 272 nm to conduct sterilization experiments against A. baumanii. We confirmed the effectiveness of ultraviolet C sterilization for both sensitive and drug resistance strains and explored the relationship between bactericidal rate and ultraviolet doses under various irradiance. Dose requirements of various irradiance were clarified. High irradiance improved sterilization efficiency greatly. The overall damage to the total genome was observed though gel electrophoresis. Ultrastructure of damaged bacteria were investigated by transmission electron microscope in detail. The study revealed that damage to DNA and to the cytoplasm matrix and ribosomes. The study has yielded the possible effects of ultraviolet light on cells by amplifying the energy. The radiation significantly promoted the production of cell wall and cellular membrane.

Significance Statement

The statistical results of bactericidal efficiency are influenced by the quantity of bacteria on the medium. Irradiance on the target surface affects the sterilization efficiency directly. And doses required in low irradiance are much more than high. A high irradiance reduced dosage efficiently to achieve the sterilization which improves the sterilization efficiency. There is a difference between low and high UVC dosage damage to the structure of bacteria. Less energy can make DNA coagulation solidified or be dispersed to the edge. Meanwhile the cytoplasm matrix is ruined. When the energy was enough, there is a boost of cell wall and cellular membrane production. The invisible light causes comprehensive damage to bacteria.
Histidine-rich protein 2: a new pathogenic factor of Plasmodium falciparum malaria
Authors: Iwasaki Takashi , Shimoda Mayu , Kanayama Haru , Kawano Tsuyoshi
Published: November 2021

Plasmodium falciparum causes serious malaria symptoms; when this protozoan parasite infects human erythrocytes, it produces and secretes large amounts of histidine-rich protein 2 (PfHRP2) into human blood. Thus, PfHRP2 is a well-known diagnostic marker for malaria infection. Here, however, we also identified PfHRP2 as a pathogenic factor produced by P. falciparum. PfHRP2 showed cell penetration and cytotoxicity against various human cells. In particular, PfHRP2 showed significant cytotoxicity over 5 days at the same concentration as in P. falciparum-infected patients’ blood (90–100 nM). This result is consistent with the mortality rate of P. falciparum malaria, which increases rapidly in untreated cases for 3–7 days. In addition, the cell penetration and cytotoxicity of PfHRP2 increased 2.5- and 2.6-fold, respectively, in the absence of serum, which suggests that low serum protein concentrations (occurring during malnutrition, for example) increase the risk of adverse effects from PfHRP2 (consistent with malnutrition increasing the lethality of malaria infection). We also showed that PfHRP2 bound to Ca2+ ions, localized to intracellular lysosomes, increased lysosomal Ca2+ levels, and inhibited the basal level of autophagy by inhibiting autolysosome formation. Furthermore, the Ca2+-dependent cytotoxicity of PfHRP2 was suppressed by the metal ion chelator ethylenediaminetetraacetic acid (EDTA). In summary, our findings suggest that PfHRP2 acts as a pathogenic factor in P. falciparum-infected patients and is associated with the exacerbation of malaria. Furthermore, EDTA is a promising candidate as a therapeutic agent for the suppression of PfHRP2 pathogenicity. Overall, this study provides new insights into P. falciparum malaria pathogenesis and treatment.
Substrate stiffness controls the cell cycle of human mesenchymal stem cells via cellular traction
Authors: Kureel Sanjay Kumar , Sinha Shatarupa , Purkayastha Purboja , Barretto Sarah , Majumder Abhijit
Published: November 2021

The microenvironment of human mesenchymal stem cells (hMSCs) regulates their self-renewal and differentiation properties. Previously it was shown that hMSCs remained quiescent on soft (0.25 kPa) polyacrylamide (PA) gels but re-entered into cell cycle on a stiff (7.5 kPa) gel. However, how cells behave on intermediate stiffness and what intracellular factors transmit mechanical changes to cell interior thereby regulating cell cycle remained unknown. In this work we demonstrated that PA gels between 1 and 5 kPa act as a mechanical switch in regulating cell cycle of hMSCs. By experiments on cell-cycle exit and re-entry, we found that hMSCs demonstrated a sharp transition from quiescence to proliferation between 1 and 5 kPa. Further studies with ROCK inhibitor Y-27632 revealed that contractile proteins, but not cell spread area, accounts for the sensitivity of hMSCs towards substrate stiffness and hence correlates with their changes in cell cycle. These observations therefore suggest that substrate stiffness regulates hMSC proliferation through contractile forces as generated by cellular contractile proteins in a unique pattern which is distinct from other cell types as studied.

Graphical abstract
Label-free imaging of collagen fibers in tissue slices using phase imaging with computational specificity
Authors: Sakakura Masayoshi , Macias Virgilia , Borhani Sohelia , Kajdacsy-Balla André , Popescu Gabriel
Published: November 2021

Evaluating the tissue collagen content in addition to the epithelial morphology has been proven to offer complementary information in histopathology, especially in oncology tumor staging and prediction of survival in cancer patients. One imaging modality widely used for this purpose is second harmonic generation microscopy (SHGM), which reports on the nonlinear susceptibility associated with the collagen fibers. Another method is polarization light microscopy (PLM) combined with picrosirius-red (PSR) tissue staining. However, SHGM requires expensive equipment and provides limited throughput, while PLM and PSR staining are not part of the routine surgical pathology workflow. Here, we utilize phase imaging with computational specificity (PICS) to computationally infer the collagen distribution ofunlabeledtissue, with high specificity. PICS utilizes deep learning to translate quantitative phase images (QPI) into corresponding PSR images with high accuracy and inference speed of 200 milisecond per forwardpass through the model once trained. We developed a multimodal imaging instrument that yields both Spatial light Inference Microscopy (SLIM) and polarized light microscopy (PLM) images from the same field of view. Our results indicate that the distributions of collagen fiber orientation, length, and straightness reported by PICS closely match the ones from ground truth as defined by KL-divergence.
Cleavage of PGAM5 by the intramembrane protease PARL is governed by transmembrane helix dynamics and oligomeric state
Authors: Siebert Verena , Silber Mara , Heuten Elena , Muhle-Goll Claudia , Lemberg Marius K.
Published: November 2021

The intramembrane protease PARL is a crucial mitochondrial safeguard by cleaving the mitophagy regulators PINK1 and PGAM5. PGAM5 substrate determinates have not been rigorously investigated and it is unclear how uncoupling the mitochondrial membrane potential regulates its processing inversely to PINK1. Here we show that in PGAM5 several hydrophilic residues distant from the cleavage site serve as key determinant for PARL- catalyzed cleavage. NMR analysis indicates that a short N-terminal amphipathic helix, followed by a kink and a C-terminal helix harboring the scissile peptide bond, is key for a productive interaction with PARL. In difference to PINK1, PGAM5 is stably inserted into the inner mitochondrial membrane until uncoupling the membrane potential triggers its disassembly into monomers that are vulnerable to PARL-catalyzed processing. We suggest a model in which PGAM5 is a slowly processed substrate with PARL-catalyzed cleavage that is influenced by multiple hierarchical substrate features including a membrane-potential- dependent oligomeric switch.
Loss of zinc transporters ZIP1 and ZIP3 augments platelet reactivity in response to G protein-coupled receptor agonists and accelerates thrombus formation in vivo
Authors: Elgheznawy A , Öftering P , Englert M , Kaiser F , Kusch C , Gbureck U , Bösl MR , Nieswandt B , Vögtle T , Hermanns HM
Published: November 2021

Zinc (Zn2+) is considered as an important mediator for thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these well-known Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly delayed and less efficient Zn2+ release upon thrombin-stimulated platelet activation. This resulted in a hyperactive platelet response not only in response to thrombin, but also towards other G protein-coupled receptor (GPCR) agonists. Immunoreceptor tyrosine-based activation (ITAM)-coupled receptor agonist signalling, however, was unaffected. Augmented GPCR responses were accompanied by enhanced Ca2+ signalling and PKC activation. Further functional analysis of ZIP1/3 double deficient mice revealed enhanced platelet aggregation, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.
Fusobacterium nucleatum induces pancreatic cancer cell proliferation and migration by regulating host autocrine and paracrine signaling
Authors: Udayasuryan Barath , Nguyen Tam T. D. , Umaña Ariana , Ahmad Raffae N. , Roberts LaDeidra Monét , Sobol Polina , Jones Stephen D. , Munson Jennifer M. , Slade Daniel J. , Verbridge Scott S.
Published: November 2021

Pancreatic ductal adenocarcinoma (PDAC) harbors a complex tumor microbiome that has been implicated in cancer progression and resistance to chemotherapy. Recent clinical investigations uncovered a correlation between high loads of intratumor Fusobacterium nucleatum and decreased patient survival. Here we show that healthy and cancerous pancreatic cell lines harboring intracellular F. nucleatum secrete increased levels of cancer-associated cytokines including GM-CSF, CXCL1, IL-8, and MIP-3α. We report that GM-CSF (granulocyte-macrophage colony stimulating factor) secretion directly increases the proliferation and migration of pancreatic cancer cells via an autocrine mechanism, notably in the absence of immune cell participation. Furthermore, we show that non-cancerous pancreatic epithelial cells do not exhibit increased proliferation or migration in response to these cytokines, but nevertheless, their secreted cytokines stimulate these responses in cancerous cell lines through paracrine signaling. Our results provide evidence that intratumor F. nucleatum in the pancreas elicits an infection-specific cytokine secretion profile from both normal and cancerous cells that adversely contributes to cancer progression through autocrine and paracrine mechanisms. Therefore, these results support the importance of investigating the contributions of both microbiome and host driven processes in pancreatic cancer to guide future therapeutic interventions.
Preclinical and Toxicology Studies of BRD5529, a Selective Inhibitor of CARD9
Authors: Kottom Theodore J. , Schaefbauer Kyle , Carmona Eva M. , Yi Eunhee S. , Limper Andrew H.
Published: November 2021

Background

Exuberant inflammation during Pneumocystis pneumonia leads to lung injury. CARD9 is a central mediator of inflammatory signaling mediated by C-type lectin receptors. CARD9 inhibitor BRD5529 has been shown to be an effective in vitro inhibitor of Pneumocystis β-glucan-induced proinflammatory signaling and downstream TNF-alpha production, suggesting its viability as a candidate for preliminary drug testing as an anti- inflammatory agent in the rodent Pneumocystis pneumonia model (PCP).

Methods

To assess for potential toxicity, mice were injected intraperitoneally (IP) daily either with vehicle or BRD5529 at 0.1 mg/kg or 1.0 mg/kg for two weeks. Mouse weights were taken daily. At day 14, mice were euthanized, weighed, and analyzed by flexiVent™ for lung stiffness. Lungs, liver, and kidney were then harvested for H&E staining and pathology scoring. Lung samples were further analyzed for proinflammatory cytokines via ELISA and extracellular matrix generation via quantitative PCR (q-PCR). Blood collection postmortem was performed for blood chemistry analysis.

Results

BRD5529 at both doses of IP administration resulted in no significant changes in daily or final weight gain. Analysis of lung stiffness by flexiVent™ showed no significant differences between the control or treated groups. Furthermore, ELISA results for proinflammatory IL-1 Beta, IL-6, and TNF-alpha showed no major differences in the respective groups. qPCR analysis of extracellular matrix transcripts collagen type I, alpha 1 (Col1a1) and fibronectin (Fn) were statically similar as well in the treated and control groups. Examination and pathology scoring of H&E slides from lung, liver, and kidney from the each of the mice in all groups and subsequent pathology scoring showed no significant change. Blood chemistry analysis revealed similar, non-significant patterns.

Conclusions

BRD5529 in our initial general safety and toxicology assessments displayed no inherent safety concerns in the analyzed parameters. These data support broader in vivo testing of the inhibitor as a timed adjunct therapy to the deleterious proinflammatory host immune response often associated with anti-Pneumocystis therapy.
IGF1 Signaling in Temporomandibular Joint Fibrocartilage Stem Cells Regulates Cartilage Growth and Homeostasis in Mice
Authors: Bi Ruiye , Luo Xueting , Li Qianli , Li Peiran , Fan Yi , Ying Binbin , Zhu Songsong
Published: November 2021

Objective

Investigate functional roles of Igf1 in fibrocartilage stem cell (FCSC) for temporomandibular joint (TMJ) cartilage growth and homeostasis.

Methods

Gli1-CreER+; RosaTdTomato mice were used for validating FCSCs lineage labeling efficiency. In Gli1-/Col2-CreER+; Igf1fl/fl mice, TMJ cartilage morphological and functional changes were characterized at 4 weeks and 5 months after Igf1 deletion. H&E, Safranine O and immuno-histochemistry staining were performed. FCSCs specificity were characterized using EdU and TUNEL staining. A unilateral anterior crossbite (UAC) mouse model was generated for mimicking TMJ osteoarthritis status.

Results

In Gli1-CreER+; RosaTdTomato mice, RFP labeled FCSCs showed favorable proliferative capacity. 4 weeks after Igf1 deletion, Gli1+ and Col2+ cell lineages led to distinct pathological changes of TMJ cartilage morphology. A more serious reduction of cartilage thickness and cell density were found in the superficial layers in Gli1-CreER+; Igf1fl/fl mice. 5 months after Igf1 deletion, more severe disordered cell arrangement in TMJ cartilage were found in both groups with Gli1+ and Col2+ specific deletion of Igf1. Immunostaining showed that PI3K/Akt signaling pathway was blocked in the superficial layers of TMJ in Gli1-CreER+; RosaTdTomato mice. Finally, deletion of Igf1 in FCSCs significantly aggravated osteoarthritis (OA) phenotypic changes in TMJ in UAC mice model, characterized in decreased cartilage thickness, cell numbers and loss of extracellular matrix secretion.

Conclusion

Igf1 deletion disrupted stem cell functions of FCSCs, leading to disordered cell distribution during TMJ growth, as well as exaggerated the OA process in TMJ under pathological condition. In TMJ cartilage, Igf1 expression in FCSCs is critical for PI3K/Akt activation, which may be involved in regulating FCSCs self-renewal and differentiation.
Nonmetric ANOVA: a generic framework for analysis of variance on dissimilarity measures
Authors: Malyutina Alina , Tang Jing , Amiryousefi Ali
Published: November 2021

Classic Analysis of Variance (ANOVA; cA) tests the explanatory power of a partitioning on a set of objects. Nonparametric ANOVA (npA) extends to a case where instead of the object values themselves, their mutual distances are available. While considerably widening the applicability of the cA, the npA does not provide a statistical framework for the cases where the mutual dissimilarity measurements between objects are nonmetric. Based on the central limit theorem (CLT), we introduce nonmetric ANOVA (nmA) as an extension of the cA and npA models where metric properties (identity, symmetry, and subadditivity) are relaxed. Our model allows any dissimilarity measures to be defined between objects where a distinctiveness of a specific partitioning imposed on those are of interest. This derivation accommodates an ANOVA-like framework of judgment, indicative of significant dispersion of the partitioned outputs in nonmetric space. We present a statistic which under the null hypothesis of no differences between the mean of the imposed partitioning, follows an exactF-distribution allowing to obtain the consequentialp-value. Three biological examples are provided and the performance of our method in relation to the cA and npA is discussed.

Significance Statement

The Nonmetric Analysis of Variance (nmANOVA) conveys a framework that allows a compatible type of ANOVA for the cases where the proper metric measurements between objects are either lost, unknown or however inaccessible. While classic ANOVA is based on the measurements of the data from a base datum, the nmANOVA is formulated on the dissimilarity outputs (not necessarily metric) defined between all objects. As the main goal of ANOVA in providing a statistical test for assessing the significance of a considered partitioning on the data, the nmANOVA is yielding a paralleled scheme of inference with 1) accommodating the outcomes dissimilarities intowithinandbetweengroups statistics, 2) assessing their respective divergence with a parametric distribution, and 3) providing a resultantp-value indicative of evidences fore rejecting the null hypothesis.
Enhancer and super-enhancer landscape in polycystic kidney disease
Authors: Lakhia Ronak , Mishra Abheepsa , Biggers Laurence , Malladi Venkat , Cobo-Stark Patricia , Hajarnis Sachin , Patel Vishal
Published: November 2021

Widespread aberrant gene expression is pathological hallmark of polycystic kidney disease (PKD). Numerous pathogenic signaling cascades, including c-Myc, Fos, and Jun are transactivated. However, the underlying epigenetic regulators are poorly defined. Here we show that H3K27ac, a histone modification that marks active enhancers, is elevated in mouse and human ADPKD samples. Using comparative H3K27ac ChIP-Seq analysis, we mapped >16000 active intronic and intergenic enhancer elements in Pkd1-mutant mouse kidneys. We find that the cystic kidney epigenetic landscape resembles that of a developing kidney, and >90% of upregulated genes in Pkd1-mutant kidneys are co-housed with activated enhancers in the same topologically associated domains. Furthermore, we identify an evolutionarily-conserved enhancer cluster downstream of the c-Myc gene and super-enhancers flanking both Jun and Fos loci in mouse and human ADPKD models. Deleting these regulatory elements reduces c-Myc, Jun, or Fos abundance and suppresses proliferation and 3D cyst growth of Pkd1-mutant cells. Finally, inhibiting glycolysis and glutaminolysis or activating Ppara in Pkd1-mutant cells lowers global H3K27ac levels and on c-Myc enhancers. Thus, our work suggests that epigenetic rewiring mediates the transcriptomic dysregulation in PKD, and the regulatory elements can be targeted to slow cyst growth.