Inferring gene regulatory networks using transcriptional profiles as dynamical attractors.

Genetic regulatory networks (GRNs) regulate the flow of genetic information from the genome to expressed messenger RNAs (mRNAs) and thus are critical to controlling the phenotypic characteristics of cells. Numerous methods exist for profiling mRNA transcript levels and identifying protein-DNA binding interactions at the genome-wide scale. These enable researchers to determine the structure and output of transcriptional regulatory networks, but uncovering the complete structure and regulatory logic of GRNs remains a challenge. The field of GRN inference aims to meet this challenge using computational modeling to derive the structure and logic of GRNs from experimental data and to encode this knowledge in Boolean networks, Bayesian networks, ordinary differential equation (ODE) models, or other modeling frameworks. However, most existing models do not incorporate dynamic transcriptional data since it has historically been less widely available in comparison to "static" transcriptional data. We report the development of an evolutionary algorithm-based ODE modeling approach (named EA) that integrates kinetic transcription data and the theory of attractor matching to infer GRN architecture and regulatory logic. Our method outperformed six leading GRN inference methods, none of which incorporate kinetic transcriptional data, in predicting regulatory connections among TFs when applied to a small-scale engineered synthetic GRN in Saccharomyces cerevisiae. Moreover, we demonstrate the potential of our method to predict unknown transcriptional profiles that would be produced upon genetic perturbation of the GRN governing a two-state cellular phenotypic switch in Candida albicans. We established an iterative refinement strategy to facilitate candidate selection for experimentation; the experimental results in turn provide validation or improvement for the model. In this way, our GRN inference approach can expedite the development of a sophisticated mathematical model that can accurately describe the structure and dynamics of the in vivo GRN.

AGB estimation using Sentinel-2 and Sentinel-1 datasets.

Climate change is one of the greatest threats recently, of which developing countries are facing most of the brunt. In the fight against climate change, forests can play an important role, since they hold a substantial amount of terrestrial carbon and can therefore affect the global carbon cycle. Deforestation, however, is a significant challenge. There are financial incentives that can help in halting deforestation by compensating developing countries for their efforts. They require however assessments which makes it essential for developing countries to regularly monitor their stocking. Based on the aforementioned, forest carbon stock assessment was conducted in Margalla Hills National Park i.e., Sub-tropical Chir Pine Forest (SCPF) and Sub-tropical Broadleaved Evergreen Forest (SBEF), in Pakistan combining field inventory with a remote-sensing-based approach using machine learning algorithms. Circular plots of a 20 m radius were used for recording the data and Sentinel-2 (S2) and Sentinel-1 (S1) satellite data were used for estimating the Aboveground Biomass (AGB). The performances of Random Forests (RF) and Support Vector Machine (SVM) were explored. The AGB was higher for the SCPF. The RF performed better for SCPF, but SVM was better for SBEF. The free available satellite data in the form of S2 and S1 data offers an advantage for AGB estimations. The combination of S2 and S1 for future AGB studies in Pakistan is also recommended.

Transcriptional control of hyphal morphogenesis in Candida albicans.

Candida albicans is a multimorphic commensal organism and opportunistic fungal pathogen in humans. A morphological switch between unicellular budding yeast and multicellular filamentous hyphal growth forms plays a vital role in the virulence of C. albicans, and this transition is regulated in response to a range of environmental cues that are encountered in distinct host niches. Many unique transcription factors contribute to the transcriptional regulatory network that integrates these distinct environmental cues and determines which phenotypic state will be expressed. These hyphal morphogenesis regulators have been extensively investigated, and represent an increasingly important focus of study, due to their central role in controlling a key C. albicans virulence attribute. This review provides a succinct summary of the transcriptional regulatory factors and environmental signals that control hyphal morphogenesis in C. albicans.

Triazine-Based Small Molecules: A Potential New Class of Compounds in the Antifungal Toolbox.

Invasive fungal infections caused by Candida species remain a significant public health problem worldwide. The increasing prevalence of drug-resistant infections and a limited arsenal of antifungal drugs underscore the need for novel interventions. Here, we screened several classes of pharmacologically active compounds against mammalian diseases for antifungal activity. We found that the synthetic triazine-based compound melanogenin (Mel) 56 is fungicidal in Candida albicans laboratory and clinical strains with minimal inhibitory concentrations of 8−16 µg/mL. Furthermore, Mel56 has general antifungal activity in several non-albicans Candida species and the non-pathogenic yeast Saccharomyces cerevisiae. Surprisingly, Mel56 inhibited the yeast-to-hyphae transition at sublethal concentrations, revealing a new role for triazine-based compounds in fungi. In human cancer cell lines, Mel56 targets the inner mitochondrial integral membrane prohibitin proteins, PHB1 and PHB2. However, Mel56 treatment did not impact C. albicans mitochondrial activity, and antifungal activity was similar in prohibitin single, double, and triple homozygous mutant strains compared to the wild-type parental strain. These results suggests that Mel56 has a novel mechanism-of-action in C. albicans. Therefore, Mel56 is a promising antifungal candidate warranting further analyses.

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&RUN Method and Data Analysis Workflow.

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations and stresses, and host-pathogen interactions. Determining the genome-wide binding of regulatory transcription factors to DNA is essential to understanding the function of transcription factors in these often complex biological processes. Cleavage under targets and release using nuclease (CUT&RUN) is a modern method for genome-wide mapping of in vivo protein-DNA binding interactions that is an attractive alternative to the traditional and widely used chromatin immunoprecipitation followed by sequencing (ChIP-seq) method. CUT&RUN is amenable to a higher-throughput experimental setup and has a substantially higher dynamic range with lower per-sample sequencing costs than ChIP-seq. Here, a comprehensive CUT&RUN protocol and accompanying data analysis workflow tailored for genome-wide analysis of transcription factor-DNA binding interactions in the human fungal pathogen Candida albicans are described. This detailed protocol includes all necessary experimental procedures, from epitope tagging of transcription factor-coding genes to library preparation for sequencing; additionally, it includes a customized computational workflow for CUT&RUN data analysis.

The challenge of antibiotic resistance in post-war Mosul, Iraq: an analysis of 20 months of microbiological samples from a tertiary orthopaedic care centre.

OBJECTIVES: Iraq has suffered unrest and conflicts in the past decades, leaving behind a weakened healthcare system. In 2018, Médecins Sans Frontières (MSF) opened a tertiary orthopaedic care centre in Mosul providing reconstructive surgery with access to microbiological analysis. METHODS: A retrospective cross-sectional analysis of microbiological and clinical data of patients admitted between April 2018 and December 2019. RESULTS: There were 174 patients who were included in this study; there were more males than females (135 to 38, respectively), and the mean age was 32.6 y. Of the 174 patients, the majority had more than one bacterial isolate detected (n = 122, 70.1%); 141 (81.0%) had at least one multidrug-resistant (MDR) isolate detected during their hospital stay. Staphylococcus aureus (n = 197, 48.2%) was the most common organism isolated. Overall, most isolates detected were MDR (n = 352, 86%), mostly methicillin-resistant S. aureus (n = 186, 52.8%) or extended-spectrum beta-lactamase-producing Enterobacterales (n = 117, 33.2%). Among patients admitted to the operating department (n = 111, 63.7%), 81.1% (n = 90) were admitted for violent trauma injuries. Patients who had more than one procedure performed per surgery had significantly increased odds of having at least one MDR organism isolated (OR 8.66, CI 1.10-68.20, P = 0.03). CONCLUSION: This study describes a high prevalence of antibiotic resistance in patients with trauma-related wounds in Mosul, Iraq. It highlights the importance of microbiological analysis and ongoing surveillance to provide optimal treatment. Additionally, it underscores the importance of infection prevention and control measures as well as antibiotic stewardship.

Neuropsychiatric Consequences of COVID-19 Pandemic: A Synthetic Review from a Global Perspective.

Some research suggests that distress, secondary to isolation and fear following COVID-19 infection, can negatively affect the long-term more than the COVID-19 infection itself. This narrative review aims to provide a global view on the neuropsychiatric consequences of COVID-19 that can be ascribed to several factors, ranging from the direct effect of infection, to the body's responses against the infection, or to the psychological sequelae of social isolation, unemployment, and fear for one's health and livelihood. Current findings show that the more severe the respiratory infection, the more likely are central nervous system (CNS) complications regarding the infection itself. The immune reactions to the infection may result in symptoms similar to chronic fatigue as well as neurocognitive deficits, which last long after the infection is gone. An increase in symptoms of depression, anxiety, and trauma-related stress may also follow upon economic fears and isolation from friends and family. The consequences of the pandemic are not limited to adults; children learning remotely and away from classmates and routine activities may develop adjustment disorders, acute stress disorder, and a variety of manifestations of grief. A summary of case reports suggests that COVID-19-related stress, economic recession, and political unrest increase the risk of suicidal behaviors and acts of violence. However, it is unknown whether manifestations of mental disorders result from social causes or whether CNS complications may be responsible.

The Roles of Chromatin Accessibility in Regulating the Candida albicans White-Opaque Phenotypic Switch.

Candida albicans, a diploid polymorphic fungus, has evolved a unique heritable epigenetic program that enables reversible phenotypic switching between two cell types, referred to as "white" and "opaque". These cell types are established and maintained by distinct transcriptional programs that lead to differences in metabolic preferences, mating competencies, cellular morphologies, responses to environmental signals, interactions with the host innate immune system, and expression of approximately 20% of genes in the genome. Transcription factors (defined as sequence specific DNA-binding proteins) that regulate the establishment and heritable maintenance of the white and opaque cell types have been a primary focus of investigation in the field; however, other factors that impact chromatin accessibility, such as histone modifying enzymes, chromatin remodelers, and histone chaperone complexes, also modulate the dynamics of the white-opaque switch and have been much less studied to date. Overall, the white-opaque switch represents an attractive and relatively "simple" model system for understanding the logic and regulatory mechanisms by which heritable cell fate decisions are determined in higher eukaryotes. Here we review recent discoveries on the roles of chromatin accessibility in regulating the C. albicans white-opaque phenotypic switch.

Prevalence of non-specific chronic low-back pain and risk factors among male soldiers in Saudi Arabia.

BACKGROUND: Non-specific chronic low back pain (NSCLBP) is the most common musculoskeletal disorder affecting health and work among the military population. NSCLBP is a complex disorder with several risk factors contributing to its occurrence. Therefore, the objective of our study was to estimate the prevalence and contribution of risk factors towards NSCLBP among male soldiers in Saudi Arabia. METHODS: A cross-sectional study was conducted from March 2020 to January 2021 among the military personnel at Hafar Al-Batin military base, Saudi Arabia. The entire population (n = 62, 000) at the military base were invited to participate in the study. The participants were invited to participate in the study either through direct referral from in-patient and out-patient departments of the military hospital or by invitation through pamphlets, email, and advertisement across the offices and residential areas. Soldiers reporting lower back pain for at least 12 weeks were screened for inclusion criteria at the physical therapy department of the military hospital. Inclusion criteria included pain or discomfort originating from the lower back without any known spinal diseases. Participants with a systemic inflammatory disorder, trauma, neurological symptoms, and recent spinal surgery were excluded. All eligible participants were assessed for demographic variables and risk factors and complete the Rolland Morris Disability Questionnaire and WHO-Five Well-Being Index. RESULTS: This study identified a 46.3% prevalence of pain originating from the spine with a 2.7% prevalence of NSCLBP. Spearman's rho correlation between the severity of disability due to NSCLBP was strongly associated with age (rs = 0.834, p < 0.01), quality of sleep (rs = 0.790, p < 0.01), body mass index (BMI) (rs = 0.617, p < 0.01), smoking (rs = 0.520, p < 0.01), co-morbidity (rs = 0.357, p < 0.01), but not with the level of physical activity (rs = 0.044, p = 0.07). CONCLUSION: There was a high prevalence of pain originating from the spine among male Saudi soldiers with a relatively low prevalence of NSCLBP. However, the prevalence of disability due to NSCLBP was strongly associated with age, sleep quality, BMI, smoking habit, and co-morbidity.

Cleavage of peptide bonds bearing ionizable amino acids at P(1) by serine proteases with hydrophobic S(1) pocket.

Enzymatic hydrolysis of the synthetic substrate succinyl-Ala-Ala-Pro-Xxx-pNA (where Xxx=Leu, Asp or Lys) catalyzed by bovine chymotrypsin (CHYM) or Streptomyces griseus protease B (SGPB) has been studied at different pH values in the pH range 3-11. The pH optima for substrates having Leu, Asp, and Lys have been found to be 7.5-8.0, 5.5-6.0, and ∼10, respectively. At the normally reported pH optimum (pH 7-8) of CHYM and SGPB, the substrate with Leu at the reactive site is more than 25,000-fold more reactive than that with Asp. However, when fully protonated, Asp is nearly as good a substrate as Leu. The pK values of the side chains of Asp and Lys in the hydrophobic S(1) pocket of CHYM and SGPB have been calculated from pH-dependent hydrolysis data and have been found to be about 9 for Asp and 7.4 and 9.7 for Lys for CHYM and SGPB, respectively. The results presented in this communication suggest a possible application of CHYM like enzymes in cleaving peptide bonds contributed by acidic amino acids between pH 5 and 6.

Highly Stretchable and Flexible Melt Spun Thermoplastic Conductive Yarns for Smart Textiles.

This study demonstrates a scalable fabrication process for producing biodegradable, highly stretchable and wearable melt spun thermoplastic polypropylene (PP), poly(lactic) acid (PLA), and composite (PP:PLA = 50:50) conductive yarns through a dip coating process. Polydopamine (PDA) treated and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) coated conductive PP, PLA, and PP/PLA yarns generated electric conductivity of 0.75 S/cm, 0.36 S/cm and 0.67 S/cm respectively. Fourier Transform Infrared Spectroscopy (FTIR) confirmed the interactions among the functional groups of PP, PLA, PP/PLA, PDA, and PEDOT:PSS. The surface morphology of thermoplastic yarns was characterized by optical microscope and Scanning Electron Microscope (SEM). The mechanical properties of yarns were also assessed, which include tensile strength (TS), Young's modulus and elongation at break (%). These highly stretchable and flexible conductive PP, PLA, and PP/PLA yarns showed elasticity of 667%, 121% and 315% respectively. The thermal behavior of yarns was evaluated by differential scanning calorimetry (DSC) and thermo-gravimetric analysis (TGA). Wash stability of conductive yarns was also measured. Furthermore, ageing effect was determined to predict the shelf life of the conductive yarns. We believe that these highly stretchable and flexible PEDOT:PSS coated conductive PP, PLA, and PP/PLA composite yarns fabricated by this process can be integrated into textiles for strain sensing to monitor the tiny movement of human motion.

CL-20 photodecomposition: ab initio foundations for identification of products.

1,5-Dihydrodiimidazo[4,5-b:4'5'e]pyrazine, 1H-imidazo[4,5-b]pyrazine, and 1H-imidazole were considered as possible products of 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) photodecomposition. Since we took as a reference the product obtained after CL-20 irradiation in methanol solution, the nature of intermolecular bonds between heterocycles under study and methanol molecules was analyzed in detail. Existing hydrogen bonds were found to be quite strong, so dependence of calculations results on an influence of solvent was taken into account using both the polarizable continuum model (PCM) and the supermolecular approach. Electronic spectra of 1,5-dihydrodiimidazo[4,5-b:4'5'e]pyrazine, 1H-imidazo[4,5-b]pyrazine and 1H-imidazole were simulated using time dependent density functional theory (TD-DFT) and single-excitation configuration interaction (CIS) method. We observed that TD-DFT excitation energies are lower if compared to corresponding values obtained by the CIS method. Results of calculations with PCM and the supermolecular approach are very close. It was found that differences between calculated gas phase excitation energies and those values obtained by applying solvent models increases when the number of conjugated bonds in a molecule increases. Oscillator strengths of UV bands of the considered molecules are higher in the gas phase than in modeled methanol solutions. We found that the predicted spectrum of 1H-imidazole is in close agreement with the experimental UV spectrum of the CL-20 photolysis product.

Application of quantum-chemical approximations to environmental problems: Prediction of physical and chemical properties of TNT and related species.

This paper presents accurate predictions of ecologically important properties of nitroaromatic compounds and their derivatives, including vapor pressure, Henry's law constants, water solubility, octanol/water partition coefficients, heats of formation and ionization potentials. The proposed technique of calculations was based on quantum-chemical methods. The relationship between the chemical structure and mentioned physico-chemical parameters of such widespread military produced contaminants as trinitrotoluene and its derivatives was considered. We revealed that the DFT level of theory combined with the COSMO-RS technique is able to predict the studied parameters with an accuracy that results in error bars of less then one logarithmic unit.

Guidance on the use of the weight of evidence approach in scientific assessments.

EFSA requested the Scientific Committee to develop a guidance document on the use of the weight of evidence approach in scientific assessments for use in all areas under EFSA's remit. The guidance document addresses the use of weight of evidence approaches in scientific assessments using both qualitative and quantitative approaches. Several case studies covering the various areas under EFSA's remit are annexed to the guidance document to illustrate the applicability of the proposed approach. Weight of evidence assessment is defined in this guidance as a process in which evidence is integrated to determine the relative support for possible answers to a question. This document considers the weight of evidence assessment as comprising three basic steps: (1) assembling the evidence into lines of evidence of similar type, (2) weighing the evidence, (3) integrating the evidence. The present document identifies reliability, relevance and consistency as three basic considerations for weighing evidence.

Effect of Osmolytes and Guanidinium Chloride on the Enzymatic Properties of Bovine α-Chymotrypsin.

We have studied the effect of guanidinium chloride (Gdn.Cl) and different osmolytes such as betaine, trimethylamine-N-oxide (TMAO) and urea on the rate of chymotrypsin catalyzed reaction. The rates were measured using three synthetic chromogenic substrates, succinyl-ala-ala-pro-arg-pNA (AAPR), succinyl-ala-ala-pro-leu-pNA (AAPL), and succinyl-ala-ala-pro-phe-pNA (AAPF). Qualitatively, the results with the three substrates were identical. Guanidinium chloride and urea produced a linear decrease while TMAO produced a linear increase in the rate with increase in osmolyte concentration. Betaine had practically no effect on the rate of enzyme catalyzed reaction up to a concentration of 1.2 M. However, quantitatively the rate change per molar concentration of osmolyte (or Gdn.Cl) was significantly larger for AAPR that has a polar and cationic reactive site residue than the two substrates (AAPL and AAPF) that have non-polar reactive site residues. These results suggest that the chemical nature of the substrate (and presumably the active site of the enzyme) plays an important role in determining the effect of osmolytes in enzyme catalyzed reactions.

Specificity of proteinase K at P2 to P3' sub-sites and its comparison to other serine proteases.

Specificity of the commercially important serine protease, proteinase K, has been investigated by measuring free energies of association of proteinase K with turkey ovomucoid third domain inhibitor variants at contact positions P2, P1, P1', P2', and P3'. Correlations of these values were run with similar values that have been obtained for six other serine proteases. Among the six proteases, subtilisin Carlsberg shows a near perfect correlation (Pearson Product correlation coefficient = 0.93 to 0.99) with proteinase K at all of these positions. Proteinase K has only 35% sequence identity with subtilisin Carlsberg, yet, the two enzymes are nearly identical in their specificity at P2 to P3' positions. With other serine proteases such as bovine chymotrypsin, human leukocyte elastase, porcine pancreatic elastase, Streptomyces griseus protease A and B, proteinase K showed relatively poor or no correlation.

Investigation of the mechanism of protein denaturation by guanidine hydrochloride-induced dissociation of inhibitor-protease complexes.

In this communication we describe an approach in which guanidine hydrochloride-induced dissociation of a protein inhibitor-serine protease complex is used to explore the molecular basis of protein denaturation. The rationale behind this approach is that the inhibitor-protease complex is stabilized by the same types of non-covalent interactions that stabilize the native state of a protein. The dissociation of inhibitor-protease complex can be performed at concentrations of guanidine hydrochloride at which the inhibitor and the protease retain their native conformations. Here, we present our results on the effect of 0.1 M to 0.4 M guanidine hydrochloride concentrations on the association equilibrium constants (reciprocal of dissociation constant) of P1G, P1A, P1V, P1N, and P1S variants of turkey ovomucoid third domain with bovine α-chymotrypsin. We use these results to calculate the free energy change in the dissociation of inhibitor-protease complexes (the m value) per mol of guanidine hydrochloride concentration. Our results agree with the general consensus that the denaturing effect of guanidine hydrochloride is due to its favorable interaction with the polar parts of proteins and that the non-polar side chains have no or little favorable interaction with guanidine hydrochloride.

Additivity-based design of the strongest possible turkey ovomucoid third domain inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB).

We describe here successful designs of strong inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB). For each enzyme two inhibitor variants were designed. In one, the reactive site residue (position 18) was retained and the best residues were substituted at contact positions 13, 14, and 15. In the other variant the best residues were substituted at all contact positions except the reactive site where a Gly was substituted. The four designed variants were: for PPE, T(13)E(14)Y(15)-OMTKY3 and T(13)E(14)Y(15)G(18)M(21)P(32)V(36)-OMTKY3, and for SGPB, S(13)D(14)Y(15)-OMTKY3 and S(13)D(14)Y(15)G(18)I(19)K(21)-OMTKY3. The free energies of association (ΔG(0)) of expressed variants have been measured with the proteases for which they were designed as well as with five other serine proteases and the results are discussed.

One-electron standard reduction potentials of nitroaromatic and cyclic nitramine explosives.

Extensive studies have been conducted in the past decades to predict the environmental abiotic and biotic redox fate of nitroaromatic and nitramine explosives. However, surprisingly little information is available on one-electron standard reduction potentials (Eo(R-NO2/R-NO2-)). The Eo(R-NO2/R-NO2-) is an essential thermodynamic parameter for predicting the rate and extent of reductive transformation for energetic residues. In this study, experimental (linear free energy relationships) and theoretical (ab initio calculation) approaches were employed to determine Eo(R-NO2/R-NO2-) for nitroaromatic, (caged) cyclic nitramine, and nitroimino explosives that are found in military installations or are emerging contaminants. The results indicate a close agreement between experimental and theoretical Eo(R-NO2/R-NO2-) and suggest a key trend: Eo(R-NO2/R-NO2-) value decreases from di- and tri-nitroaromatic (e.g., 2,4-dinitroanisole) to nitramine (e.g., RDX) to nitroimino compound (e.g., nitroguanidine). The observed trend in Eo(R-NO2/R-NO2-) agrees with reported rate trends for reductive degradation, suggesting a thermodynamic control on the reduction rate under anoxic/suboxic conditions.