Structural and mechanistic insights into ALS patient derived mutations in D-amino acid oxidase.

The D-amino acid oxidase protein modulates neurotransmission by controlling the levels of D-serine, a co-agonist of N-methyl-D-aspartate receptors. Mutations in the DAO gene have been associated with ALS, with some studies reporting pathogenic mechanisms of the R199W mutation. We have characterized two novel mutations R38H and Q201R found in ALS patients and report certain novel findings related to the R199W mutation. We report the first instance of crystal structure analysis of a patient-derived mutant of DAO, R38H, solved at 2.10 Å. The structure revealed significant perturbations and altered binding with the cofactor (FAD) and the inhibitor benzoate, supported by biochemical assays. Q201R-DAO also exhibited significantly lower ligand binding efficiency. Furthermore, kinetic analysis across all variants revealed reduced oxidase activity and substrate binding. Notably, R38H-DAO exhibited near-WT activity only at high substrate concentrations, while R199W-DAO and Q201R-DAO displayed drastic activity reduction. Additionally, structural perturbations were inferred for R199W-DAO and Q201R-DAO, evident by the higher oligomeric state in the holoenzyme form. We also observed thermal instability in case of R199W-DAO mutant. We hypothesize that the mutant enzymes may be rendered non-functional in a cellular context, potentially leading to NMDAR-associated excitotoxicity. The study provides novel insights into structural and functional aspects of DAO mutations in ALS.

Aggregation of E121K mutant D-amino acid oxidase and ubiquitination-mediated autophagy mechanisms leading to amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a terminal adult-onset neuromuscular disorder. Our group has been studying this illness and previously reported novel mutations and rare mutations in a study using next-generation sequencing of DNA samples from Indian ALS patients. In this paper, we focus on the E121K mutation in the DAO gene to understand how it leads to ALS. Our experiments in SH-SY5Y cells indicate that the E121K mutation results in the accumulation of mutant protein aggregates, a change in cell morphology, and the death of neuronal cells. These protein aggregates get ubiquitinated and cause an imbalance in autophagy regulation. We observed an increase in the cellular concentrations of p62, OPTN, and LC3II. Through confocal microscopy studies, we show that the binding of p62 with ubiquitinated aggregates and its recruitment to LC3II mediates autophagosome generation. These relative changes in the key partners in autophagy increase cell death in cells harboring the E121K mutation and is a probable mechanism leading to ALS.

Barriers and facilitators for interventions to improve ART adherence in Sub-Saharan African countries: A systematic review and meta-analysis.

BACKGROUND: The HIV/AIDS pandemic remains a significant public health issue, with sub-Saharan Africa (SSA) at its epicentre. Although antiretroviral therapy (ART) has been introduced to decrease new infections and deaths, SSA reports the highest incidence of HIV/AIDS, constituting two-thirds of the global new infections. This review aimed to elucidate the predominant barriers and facilitators influencing ART adherence and to identify effective strategies to enhance ART adherence across SSA. METHODS: A comprehensive review was conducted on studies examining barriers to ART adherence and interventions to boost adherence among HIV-positive adults aged 15 and above in SSA, published from January 2010 onwards. The research utilized databases like Medline Ovid, CINAHL, Embase, and Scopus. Included were experimental and quasi-experimental studies, randomized and non-randomized controlled trials, comparative before and after studies, and observational studies such as cross-sectional, cohort, prospective and retrospective studies. Two independent reviewers screened the articles, extracted pertinent data, and evaluated the studies' methodological integrity using Joanna Briggs Institute's standardized appraisal tools. The compiled data underwent both meta-analysis and narrative synthesis. RESULTS: From an initial pool of 12,538 papers, 45 were selected (30 for narrative synthesis and 15 for meta-analysis). The identified barriers and facilitators to ART adherence were categorized into seven principal factors: patient-related, health system-related, medication-related, stigma, poor mental health, socioeconomic and socio-cultural-related factors. Noteworthy interventions enhancing ART adherence encompassed counselling, incentives, mobile phone short message service (SMS), peer delivered behavioural intervention, community ART delivery intervention, electronic adherence service monitoring device, lay health worker lead group intervention and food assistance. The meta-analysis revealed a statistically significant difference in ART adherence between the intervention and control groups (pooled OR = 1.56, 95%CI:1.35-1.80, p = <0.01), with evidence of low none statistically significant heterogeneity between studies (I2 = 0%, p = 0.49). CONCLUSION: ART adherence in SSA is influenced by seven key factors. Multiple interventions, either standalone or combined, have shown effectiveness in enhancing ART adherence. To optimize ART's impact and mitigate HIV's prevalence in SSA, stakeholders must consider these barriers, facilitators, and interventions when formulating policies or treatment modalities. For sustained positive ART outcomes, future research should target specific underrepresented groups like HIV-infected children, adolescents, and pregnant women in SSA to further delve into the barriers, facilitators and interventions promoting ART adherence.

Role of ALS-associated OPTN-K489E mutation in neuronal cell-death regulation.

Optineurin (OPTN) gene is a marker of amyotrophic lateral sclerosis (ALS). However, the role of optineurin protein (OPTN) in ALS pathology is unclear, even though it is known to regulate autophagy, apoptosis, and other survival-death cellular processes. Genetic analysis of Indian ALS patients by our group ascertained a novel mutation K489E in the OPTN gene. To identify the molecular mechanism associated with OPTN and its mutation, we developed an in-vitro cell model using SH-SY5Y cells harbouring OPTN and OPTN-K489E mutation along with its control vector. Since we observed a significant decrease in cell viability in the mutant, we measured the expressions of genes and proteins mediating apoptosis, necroptosis, and autophagy, to establish the role of OPTN in cell death regulation. Our results show that OPTN-K489E mutation changes the relative gene expressions of miRNA-9, REST, CoREST and BDNF, and causes apoptosis. We also observed an up-regulation in the expressions of necroptosis mediated genes RIPK1, RIPK3, and MLKL and autophagy mediated genes TBK1, P62, and LC3II. The results of FACS analyses revealed that this mutation promotes apoptotic and necroptotic processes confirming the pathogenicity of OPTN-K489E.

Barriers and facilitators to ART adherence among ART non-adherence people living with HIV in Cameroon: A qualitative phenomenological study.

BACKGROUND: Antiretroviral therapy (ART) needs to be taken for life with near perfect levels of adherence for it to be effective. Nonetheless, ART non-adherence is still observed in sub-Saharan African (SSA) countries such as Cameroon. The objective of this study was to assess the factors influencing non-adherence and or adherence among people living with HIV (PLWH) who have experienced non-adherence to ART in Cameroon. METHODS: A descriptive qualitative study of PLWH who have experienced non-adherence with ART in Cameroon was conducted. Data were collected using in-depth interviews. Collected data were analyzed using the NVIVO 12 software. RESULTS: In total, 43 participants participated in this study. The Southwest and Littoral regions each contributed 15 (34.88%) of participants, participants' mean age was 37.1 years (SD: 9.81) and majority 34 (82.93%) were females. ART adherence barriers include those related to patient (forgetfulness, business with other things, unwillingness to swallow drugs daily), medication (side effects), health service (arrogance of caregivers, occasional drug shortages at treatment centre, poor counseling of patient), stigma (fear of status disclosure), use of alternative treatment (traditional medicine, prayers and deliverance), resource limitation (limited food, limited finances), environmental/social (limited or no home support), and political instability (disruption of free circulation by ghost towns, roadblocks and gunshots in some regions). ART adherence facilitators include social support (family and peer support), aligning treatment with patient's daily routines (align ART with schedule of family members), use of reminders (phone alarm, sound of church bell), health sector/caregiver support (messages to patient, financial support, proper counseling), and patient's awareness of HIV status/ART knowledge (awareness of HIV positive status, Knowledge of ART benefits). CONCLUSION: ART adherence barriers in Cameroon include those related to patient, medication, health service, stigma, use of alternative treatment, resource limitation, environmental/social, and political instability. ART adherence facilitators include social support, aligning treatment with patient's daily routines, use of reminders, health sector/caregiver support, and patient's awareness of HIV status/ART knowledge. Given these barriers and facilitators, continuous information provision and consistent support both from patients' families and caregivers are needed to improve adherence among patients. Further studies including many regions and larger samples using both in-depth and focused group discussions as well as quantitative approaches are required to uncover the burden related to ART non-adherence.

Characterization of E121K mutation of D-amino acid oxidase - Insights into mechanisms leading to amyotrophic lateral sclerosis.

D-amino acid oxidase (DAO) maintains the intracellular d-serine level which modulates the activity of the N-methyl-d-aspartate receptor and its dysfunction has been linked to several neurodegenerative disorders. In targeted next-generation sequencing study by our group, E121K mutation in DAO was associated with amyotrophic lateral sclerosis (ALS) in patients from India. However, variations in molecular mechanisms caused by this mutation which leads to ALS have not been studied. Hence, we carried out comparative biophysical characterization and assay studies of the wildtype- and mutant E121K-DAO. We observed that the purified E121K-DAO was inactive and exhibited a lower affinity for the FAD cofactor and benzoate inhibitor. Structural studies revealed that the E121K mutant has higher beta-sheet content, melting temperature, and oligomeric states compared to the wildtype. Kinetic study of aggregation of the variants using thioflavin-T confirmed that the E121K-DAO was more prone to aggregation. Microscopic visualization showed that the aggregation proceeds through an intermediate step involving the formation of fibrillar structures in the E121K mutant. Our results give insights into the underlying mechanisms leading to ALS pathogenesis.

Prevalence and factors associated with HIV treatment non-adherence among people living with HIV in three regions of Cameroon: A cross-sectional study.

BACKGROUND: In Cameroon, HIV care decentralization is enforced as a national policy, but follow-up of people living with HIV (PLWH) is provider-driven, with little patient education and limited patient participation in clinical surveillance. These types of services can result in low antiretroviral therapy (ART) adherence. The objective of this study was to assess the prevalence and predictors of ART non-adherence among PLWH in Cameroon. METHODS: A cross-sectional descriptive study of PLWH in HIV treatment centres in Cameroon was conducted. Only PLWH, receiving treatment in a treatment centre within the country, who had been on treatment for at least six months and who were at least 21 years old were included in the study. Individuals were interviewed about their demographics and ART experiences. Data were collected using a structured interviewer-administered questionnaire and analyzed using STATA version 14. RESULTS: A total of 451 participants participated in this study, 33.48% were from the country's Southwest region. Their mean age was 43.42 years (SD: 10.42), majority (68.89%) were females. Overall proportion of ART non-adherence among participants was 37.78%, 35.88% missed taking ART twice in the last month. Reasons for missing ART include forgetfulness, business and traveling without drugs. Over half of participants (54.67%) know ART is life-long, 53.88% have missed ART service appointments, 7.32% disbelieve in ART benefits, 28.60% think taking ART gives unwanted HIV Status reminder and 2.00% experienced discrimination seeking ART services. In the multivariate analysis, odds of ART non-adherence in participants aged 41 and above was 0.35 times (95%CI: 0.14, 0.85) that in participants aged 21-30 years, odds of ART non-adherence comparing participants who attained only primary education to those who attained higher than secondary education was 0.57 times (95%CI: 0.33, 0.97) and the odds of ART non-adherence in participants who are nonalcohol consumers was 0.62 times (95%CI: 0.39, 0.98) that in alcohol consumers. CONCLUSION: High proportion of participants are ART non-adherent, and the factors significantly associated with ART non-adherence include age, education and alcohol consumption. However, some reasons for missing ART are masked in participants' limited knowledge in taking ART, disbelief in ART benefits, feelings that ART gives unwanted HIV status reminder and experiencing discrimination when seeking ART services. These underscores need to improve staff (health personnel) attitudes, staff-patient-communication, and proper ART prior initiation counselling of patients. Future studies need to focus on assessing long-term ART non-adherence trends and predictors using larger samples in many treatment centres and regions.

Advances in bioreactor control for production of biotherapeutic products.

Advanced control strategies are well established in chemical, pharmaceutical, and food processing industries. Over the past decade, the application of these strategies is being explored for control of bioreactors for manufacturing of biotherapeutics. Most of the industrial bioreactor control strategies apply classical control techniques, with the control system designed for the facility at hand. However, with the recent progress in sensors, machinery, and industrial internet of things, and advancements in deeper understanding of the biological processes, coupled with the requirement of flexible production, the need to develop a robust and advanced process control system that can ease process intensification has emerged. This has further fuelled the development of advanced monitoring approaches, modeling techniques, process analytical technologies, and soft sensors. It is seen that proper application of these concepts can significantly improve bioreactor process performance, productivity, and reproducibility. This review is on the recent advancements in bioreactor control and its related aspects along with the associated challenges. This study also offers an insight into the future prospects for development of control strategies that can be designed for industrial-scale production of biotherapeutic products.

Residents' perception and worldview about radon control policy in Canada: A pro-equity social justice lens.

Radon is a potent indoor air pollutant, especially in radon prone areas and in countries with long winters. As the second top lung carcinogen, radon is disproportionately affecting certain population subgroups. While many provinces have taken sporadic actions, the equity issue has remained unaddressed across all policy measures. Attempts to enforce radon guidelines and enact building regulations without considering residents' views have proved ineffective. Research linking residents' radon risk perception and worldviews regarding radon control policy is lacking in Canada. We applied mixed (quantitative and qualitative) methods in a pro-equity social justice lens to examine the variations in residents' risk perception, access to risk communication messages, and worldviews about risk management across the sociodemographic strata. Triangulation of the quantitative and qualitative findings strengthened the evidence base to identify challenges and potential solutions in addressing the health risk through upstream policy actions. Enacting radon control policy requires actions from all levels of governments and relevant stakeholders to ensure equal opportunities for all residents to take the preventive and adaptive measures. Small sample size limited the scope of findings for generalization. Future studies can examine the differential impacts of radon health risk as are determined by various sociodemographic variables in a representative national cohort.

In vitro toxicity screening of amorphous silica nanoparticles using mitochondrial fraction exposure followed by MS-based proteomic analysis.

Silica nanoparticles (SiNPs) are used in consumer products, engineering and medical technologies. Attractive properties of SiNPs (e.g. size/surface-modification) enhance usage and thus the likelihood of environmental/human exposures. The assessment of health risks associated with exposures to SiNPs requires information on their relative potencies and toxicity mechanisms. In this work, phagocytic J774 cells were exposed to amorphous pristine (15, 30, 75 nm) and surface-modified (-NH2, -C3COOH, -C11COOH, -PEG) SiNP variants, and internalization was assessed by transmission electron microscopy (TEM), while cellular ATP was measured as a cytotoxicity endpoint. Furthermore, mitochondrial fractions from J774 cells were exposed to these SiNP variants (5, 15 µg mL-1), as well as two reference particles (SiNP 12 nm and TiO2), and proteomic changes were analyzed by mass spectrometry. Ingenuity Pathway Analysis was used to identify toxicity pathways. TEM analyses showed SiNP internalization and distribution along with some changes in mitochondrial structure. SiNP size- and surface-modification and chemical composition-related changes in mitochondrial proteins, including key proteins of the respiratory complex and oxidative stress, were evident based on high content mass spectrometry data. In addition, the dose-related decrease in cellular ATP levels in SiNP-exposed cells was consistent with related mitochondrial protein profiles. These findings suggest that physicochemical properties can be determinants of SiNP exposure-related mitochondrial effects, and mitochondrial exposures combined with proteomic analysis can be valuable as a new approach methodology in the toxicity screening of SiNPs for risk assessment, with added insight into related toxicity mechanisms.

Adherence barriers and interventions to improve ART adherence in Sub-Saharan African countries: A systematic review protocol.

BACKGROUND: The HIV/AIDS pandemic continues to be a major public health concern, particularly in Sub-Saharan Africa (SSA). Despite efforts to reduce new infections and deaths with the use of antiretroviral therapy (ART), SSA countries continue to bear the heaviest burden of HIV/AIDS globally, accounting for two-thirds of global new infections. The goal of this review is to identify common barriers to ART adherence as well as common effective interventions that can be implemented across SSA countries to improve ART adherence. METHODS: A systematic review of published studies on adult HIV-positive patients aged 15 or above, that have assessed the barriers to ART adherence and interventions improving patients' adherence to ART in SSA countries shall be conducted. We will conduct electronic searches for articles that have been published starting from January 2010 onwards. The databases that shall be searched will include Medline Ovid, CINAHL, Embase, and Scopus. The review will include experimental and quasi-experimental studies such as randomized and non-randomized controlled trials as well as comparative before and after studies, and observational studies-cross-sectional studies, cohort studies, prospective and retrospective studies. Two independent reviewers will screen all identified studies, extract data and appraise the methodological quality of the studies using standard critical appraisal tools from the Joanna Briggs Institute. The extracted data will be subjected to a meta-analysis and narrative synthesis. DISCUSSION: This review will synthesize existing evidence on ART adherence barriers and strategies for improving patient adherence to ART in SSA countries. It will identify common barriers to adherence and common interventions proven to improve adherence across SSA. We anticipate that the findings of this review will provide information policy makers and stakeholders involved in the fight against HIV, will find useful in deriving better ways of not only retaining patients on treatment but having them adhere to their treatment. REVIEW REGISTRATION: This protocol has been registered with the International Prospective Register of Systematic Reviews (PROSPERO); registration number CRD42021262256.

A distant angiogenin variant causes amyotrophic lateral sclerosis through loss-of-function mechanisms: Insights from long-timescale atomistic simulations and conformational dynamics.

Amyotrophic Lateral Sclerosis (ALS) is a progressive and incurable neurodegenerative disorder characterized by the degeneration of motor neurons leading to severe muscle atrophy, respiratory failure and death within 3-5 years of disease onset. Missense mutations in Angiogenin (ANG) cause ALS through loss of either ribonucleolytic activity or nuclear translocation activity or both of these functions. Although loss-of-function mechanisms of several rare and ALS-causing ANG variants have been studied before, the structure-function relationship and subsequent functional loss mechanisms of certain novel and uncharacterized rare variants have not been deciphered hitherto. In this study, the structural and dynamic properties of the distantly-located I71V variant, on the functional sites of ANG have been investigated to understand its role in ALS etiology and progression. The I71V variant has a minor allele frequency of <0.06% and thus is classified as a rare variant. Our extensive in silico investigation comprising 1-µs molecular dynamics (MD) simulations, conformational dynamics and related integrated analyses reveal that the I71V variant induces a characteristic conformational switching of catalytic His114 residue resulting in loss of ribonucleolytic activity. Molecular docking and a residue-residue interaction network propagated by an allosteric pathway further support these findings. Moreover, while no conformational alteration of nuclear localization signal governing the nuclear translocation activity was observed, an escalation in mutant plasticity was detected in the structural and essential dynamics simulations. Overall, our study emphasizes that the structure-function relationship of frequently mutating novel ANG variants needs to be established and prioritized in order to advance the pathophysiology and therapeutics of ALS.

Electron microscopy-based semi-automated characterization of aggregation in monoclonal antibody products.

Aggregation is a critical parameter for protein-based therapeutics, due to its impact on the immunogenicity of the product. The traditional approach towards characterization of such products is to use a collection of orthogonal tools. However, the fact that none of these tools is able to completely classify the distribution and physical characteristics of aggregates, implies that there exists a need for additional analytical methods. We report one such method for characterization of heterogeneous population of proteins using transmission electron microscopy. The method involves semi-automated, size-based clustering of different protein species from micrographs. This method can be utilized for quantitative characterization of heterogeneous populations of antibody/protein aggregates from TEM images of proteins, and may also be applicable towards other instances of protein aggregation.

Neuropathogenesis of SARS-CoV-2 infection.

The COVID-19 pandemic caused by the SARS-CoV-2 has recently emerged as a serious jolt to human life and economy. Initial knowledge established pulmonary complications as the chief symptom, however, the neurological aspect of the disease is also becoming increasingly evident. Emerging reports of encephalopathies and similar ailments with the detection of the virus in the CSF has elicited an urgent need for investigating the possibility of neuroinvasiveness of the virus, which cannot be ruled out given the expression of low levels of ACE2 receptors in the brain. Sensory impairments of the olfactory and gustatory systems have also been reported in a large proportion of the cases, indicating the involvement of the peripheral nervous system. Hence, the possibility of neurological damage caused by the virus demands immediate attention and investigation of the mechanisms involved, so as to customize the treatment of patients presenting with neurological complications.

Acellular oxidative potential assay for screening of amorphous silica nanoparticles.

Silica nanoparticles (SiNPs) are used in a wide range of consumer products, engineering and medical applications, with likelihood of human exposure and potential health concerns. It is essential to generate toxicity information on SiNP forms and associated physicochemical determinants to conduct risk assessment on these new materials. To address this knowledge gap, we screened a panel of custom synthesized, well-characterized amorphous SiNPs pristine and surface-modified (-C3-COOH, -C11-COOH, -NH2, -PEG) of 5 different sizes: (15, 30, 50, 75, 100 nm) for their oxidative potential using an acellular assay. The assay is based on oxidation of dithiothreitol (DTT) by reactive oxygen species and can serve as a surrogate test for oxidative stress. These materials were characterized for size distribution, aggregation, crystallinity, surface area, surface modification, surface charge and metal content. Tests for association between oxidative potential of SiNPs and their physicochemical properties were carried out using analysis of variance and correlation analyses. These test results suggest that the size of amorphous SiNPs influenced their oxidative potential irrespective of the surface modification, with 15 nm exhibiting relatively higher oxidative potential compared to the other sizes. Furthermore, SiNP surface area, surface modification and agglomeration in solution also appeared to affect oxidative potential of these SiNPs. These findings indicate that physicochemical properties are critical in influencing the oxidative behaviour of amorphous SiNPs, with potential to trigger cellular oxidative stress and thus toxicity, when exposed. This information advances our understanding of potential toxicities of these amorphous SiNPs and supports risk assessment efforts and the design of safer forms of silica nanomaterials.

Identification and characterization of novel and rare susceptible variants in Indian amyotrophic lateral sclerosis patients.

Rare missense variants play a crucial role in amyotrophic lateral sclerosis (ALS) pathophysiology. We report rare/novel missense variants from 154 Indian ALS patients, identified through targeted sequencing of 25 ALS-associated genes. As pathogenic variants could explain only a small percentage of ALS pathophysiology in our cohort, we investigated the frequency of tolerated and benign novel/rare variants, which could be potentially ALS susceptible. These variants were identified in 5.36% (8/149) of sporadic ALS (sALS) cases; with one novel variant each in ERBB4, SETX, DCTN1, and MATR3; four rare variants, one each in PON2 and ANG and two different rare variants in SETX. Identified variants were either absent or present at extremely rare frequencies (MAF < 0.01) in large population databases and were absent in 50 healthy controls sequenced through Sanger method. Furthermore, an oligogenic basis of ALS was observed in three sALS, with co-occurrence of intermediate-length repeat expansions in ATXN2 and a rare/novel variant in DCTN1 and SETX genes. Additionally, molecular dynamics and biochemical functional analysis of an angiogenin variant (R21G) identified from our cohort demonstrated loss of ribonucleolytic and nuclear translocation activities. Our findings suggest that rare variants could be potentially pathogenic and functional studies are warranted to decisively establish the pathogenic mechanisms associated with them.

Rare Angiogenin and Ribonuclease 4 variants associated with amyotrophic lateral sclerosis exhibit loss-of-function: a comprehensive in silico study.

Amyotrophic Lateral Sclerosis (ALS), a debilitating neurodegenerative disorder is related to mutations in a number of genes, and certain genes of the Ribonuclease (RNASE) superfamily trigger ALS more frequently. Even though missense mutations in Angiogenin (ANG) and Ribonuclease 4 (RNASE4) have been previously shown to cause ALS through loss-of-function mechanisms, understanding the role of rare variants with a plausible explanation of their functional loss mechanisms is an important mission. The study aims to understand if any of the rare ANG and RNASE4 variants catalogued in Project MinE consortium caused ALS due to loss of ribonucleolytic or nuclear translocation or both these activities. Several in silico analyses in combination with extensive molecular dynamics (MD) simulations were performed on wild-type ANG and RNASE4, along with six rare variants (T11S-ANG, R122H-ANG, D2E-RNASE4, N26K-RNASE4, T79A-RNASE4 and G119S-RNASE4) to study the structural and dynamic changes in the catalytic triad and nuclear localization signal residues responsible for ribonucleolytic and nuclear translocation activities respectively. Our comprehensive analyses comprising 1.2 µs simulations with a focus on physicochemical, structural and dynamic properties reveal that T11S-ANG, N26K-RNASE4 and T79A-RNASE4 variants would result in loss of ribonucleolytic activity due to conformational switching of catalytic His114 and His116 respectively but none of the variants would lose their nuclear translocation activity. Our study not only highlights the importance of rare variants but also demonstrates that elucidating the structure-function relationship of mutant effectors is crucial to gain insights into ALS pathophysiology and in developing effective therapeutics.

Implementing Process Analytical Technology for the Production of Recombinant Proteins in Escherichia coli Using an Advanced Controller Scheme.

The performance of a bioreactor in meeting process goals is affected by the microorganism used, medium composition, and operating conditions. A typical bioreactor uses a supervisory control and data acquisition (SCADA) system for control, and a combination of software and hardware tools for real-time data analysis. However, when the process is disrupted by utility or instrumentation failure, typical process controllers may be unable to reinstate normal operating conditions before the cells in the reactor shift to unfavorable metabolic regimes. The objective of this study is to examine how the response of a controller affects process recovery when disruptive incidences occur under a process analytical technology (PAT) framework. The process used for this investigation is the production of lethal toxin-neutralizing factor (LTNF) by Escherichia coli (E. coli), which is controlled by a decoupled input-output-linearizing controller (DIOLC). The performance of the DIOLC is compared to a proportional integral derivative (PID) controller subjected to the same conditions. The disruptions are introduced manually and the effect of controller action on process recovery and LTNF synthesis is measured in terms of peak purity and concentration. It is observed that DIOLC performs better after reinstating operating conditions and results in a meaningful improvement in performance.

Radon interventions around the globe: A systematic review.

BACKGROUND: Radon is the primary source of environmental radiation exposure posing a significant human health risk in cold countries. In Canada, most provinces have revised building codes by 2017, requiring construction solutions to avoid radon in all new buildings. While various construction solutions and remediation techniques have been proposed and evaluated, the question about the best method that would effectively reduce radon in a variety of contexts remained unanswered. Radon practitioners, officials of radon control programs, and businesses offering radon testing and mitigation services, builders, property managers, homeowners and residents also have similar queries. OBJECTIVE: This paper systematically reviewed both experimental and observational studies (S) with radon interventions (I) used globally in residential houses (P) compared to other residential or model houses (C) to evaluate relative mitigation effectiveness (O) that could guide selecting the best radon reduction strategy for residential buildings. METHODS: Two researchers searched fifteen academic bibliographic and grey literature databases for radon intervention studies conducted around the world, with particular emphasis on areas of North America and Europe published from 1990 to 2018.Interventions in residential and model houses were included, but studies piloted purely in the lab were excluded; the PRISMA checklist was used to synthesize data; Cochrane and Hamilton tools were used to evaluate study quality. RESULTS: Studies around the globe have investigated a variety of construction solutions, radon mitigation and remediation systems with different levels of effectiveness. In most cases, sub-slab or sump depressurization system (SSDS) with active ventilation technique was found more effective in achieving a significant and sustained radon reduction than the passive methods such as sealing, membrane, block and beam, simple ventilation, or filtration. The choice of an optimal strategy largely depends on the factors related to the initial radon level, routes of entry, building design and age, as well as other geologic, atmospheric, and climatic conditions. CONCLUSION: Although an active SSDS is the best mitigation systems, at places, it needs to be combined with another system and installed by a trained radon professional considering the pertinent factors to ensure radon level continues to remain below the action level. This study did not conduct any economic evaluation of the mitigation measures. Future review with studies on the implementation of new building codes will provide updated evidence. RECOMMENDATION: For the practical implementation of radon mitigation, training of the construction industry, information provision for residents, the establishment of public funds, incorporation of radon-prone areas in the land utilization maps, and enacting building codes deemed essential.

Immune Differentiation Regulator p100 Tunes NF-κB Responses to TNF.

Tumor necrosis factor (TNF) is a pleiotropic cytokine whose primary physiological function involves coordinating inflammatory and adaptive immune responses. However, uncontrolled TNF signaling causes aberrant inflammation and has been implicated in several human ailments. Therefore, an understanding of the molecular mechanisms underlying dynamical and gene controls of TNF signaling bear significance for human health. As such, TNF engages the canonical nuclear factor kappa B (NF-κB) pathway to activate RelA:p50 heterodimers, which induce expression of specific immune response genes. Brief and chronic TNF stimulation produces transient and long-lasting NF-κB activities, respectively. Negative feedback regulators of the canonical pathway, including IκBα, are thought to ensure transient RelA:p50 responses to short-lived TNF signals. The non-canonical NF-κB pathway mediates RelB activity during immune differentiation involving p100. We uncovered an unexpected role of p100 in TNF signaling. Brief TNF stimulation of p100-deficient cells triggered an additional late NF-κB activity consisting of RelB:p50 heterodimers, which modified the TNF-induced gene-expression program. In p100-deficient cells subjected to brief TNF stimulation, RelB:p50 not only sustained the expression of a subset of RelA-target immune response genes but also activated additional genes that were not normally induced by TNF in WT mouse embryonic fibroblasts (MEFs) and were related to immune differentiation and metabolic processes. Despite this RelB-mediated distinct gene control, however, RelA and RelB bound to mostly overlapping chromatin sites in p100-deficient cells. Repeated TNF pulses strengthened this RelB:p50 activity, which was supported by NF-κB-driven RelB synthesis. Finally, brief TNF stimulation elicited late-acting expressions of NF-κB target pro-survival genes in p100-deficient myeloma cells. In sum, our study suggests that the immune-differentiation regulator p100 enforces specificity of TNF signaling and that varied p100 levels may provide for modifying TNF responses in diverse physiological and pathological settings.