Behavioral Lifestyle Interventions for Weight Loss in Overweight or Obese Patients with Type 2 Diabetes: A Systematic Review of the Literature.

PURPOSE OF REVIEW: Around 80-90% of patients with type 2 diabetes mellitus (T2DM) are overweight or obese, presenting a greater risk for serious health complications and mortality. Thus, weight loss represents a main goal for T2DM management. Although behavioral lifestyle interventions (BLIs) could help promoting weight loss in T2DM patients with overweight or obesity, their effectiveness is still controversial. This systematic review offers an updated and comprehensive picture of BLIs according to Michie's classification in T2DM patients with overweight or obesity and identifies possible factors (related to both patients and interventions) associated with weight loss. The PRISMA guidelines were followed. The literature search till March 2023 indicated 31 studies involving 42 different BLIs. RECENT FINDINGS: Our findings suggest that structured BLIs, characterized by frequent feedback and support, can lead to a clinically meaningful 5% weight loss, regardless of specific behavioral, diet, and physical activity components. Further research should address methodological issues and heterogeneity of interventions, also considering the effect of pharmacological therapies on weight reduction. Lastly, more attention should be paid to the long-term effectiveness of behavioral lifestyle interventions and to the relationship between weight loss and diabetes.

Precision RNAi using synthetic shRNAmir target sites.

Loss-of-function genetic tools are widely applied for validating therapeutic targets, but their utility remains limited by incomplete on- and uncontrolled off-target effects. We describe artificial RNA interference (ARTi) based on synthetic, ultra-potent, off-target-free shRNAs that enable efficient and inducible suppression of any gene upon introduction of a synthetic target sequence into non-coding transcript regions. ARTi establishes a scalable loss-of-function tool with full control over on- and off-target effects.

Application of Droplet Digital PCR Technology in Muscular Dystrophies Research.

Although they are considered rare disorders, muscular dystrophies have a strong impact on people's health. Increased disease severity with age, frequently accompanied by the loss of ability to walk in some people, and the lack of treatment, have directed the researchers towards the development of more effective therapeutic strategies aimed to improve the quality of life and life expectancy, slow down the progression, and delay the onset or convert a severe phenotype into a milder one. Improved understanding of the complex pathology of these diseases together with the tremendous advances in molecular biology technologies has led to personalized therapeutic procedures. Different approaches that are currently under extensive investigation require more efficient, sensitive, and less invasive methods. Due to its remarkable analytical sensitivity, droplet digital PCR has become a promising tool for accurate measurement of biomarkers that monitor disease progression and quantification of various therapeutic efficiency and can be considered a tool for non-invasive prenatal diagnosis and newborn screening. Here, we summarize the recent applications of droplet digital PCR in muscular dystrophy research and discuss the factors that should be considered to get the best performance with this technology.

Interrogation of cancer gene dependencies reveals paralog interactions of autosome and sex chromosome-encoded genes.

Genetic networks are characterized by extensive buffering. During tumor evolution, disruption of functional redundancies can create de novo vulnerabilities that are specific to cancer cells. Here, we systematically search for cancer-relevant paralog interactions using CRISPR screens and publicly available loss-of-function datasets. Our analysis reveals >2,000 candidate dependencies, several of which we validate experimentally, including CSTF2-CSTF2T, DNAJC15-DNAJC19, FAM50A-FAM50B, and RPP25-RPP25L. We provide evidence that RPP25L can physically and functionally compensate for the absence of RPP25 as a member of the RNase P/MRP complexes in tRNA processing. Our analysis also reveals unexpected redundancies between sex chromosome genes. We show that chrX- and chrY-encoded paralogs, such as ZFX-ZFY, DDX3X-DDX3Y, and EIF1AX-EIF1AY, are functionally linked. Tumor cell lines from male patients with loss of chromosome Y become dependent on the chrX-encoded gene. We propose targeting of chrX-encoded paralogs as a general therapeutic strategy for human tumors that have lost the Y chromosome.

Dietary Phytoestrogens and Their Metabolites as Epigenetic Modulators with Impact on Human Health.

The impact of dietary phytoestrogens on human health has been a topic of continuous debate since their discovery. Nowadays, based on their presumptive beneficial effects, the amount of phytoestrogens consumed in the daily diet has increased considerably worldwide. Thus, there is a growing need for scientific data regarding their mode of action in the human body. Recently, new insights of phytoestrogens' bioavailability and metabolism have demonstrated an inter-and intra-population heterogeneity of final metabolites' production. In addition, the phytoestrogens may have the ability to modulate epigenetic mechanisms that control gene expression. This review highlights the complexity and particularity of the metabolism of each class of phytoestrogens, pointing out the diversity of their bioactive gut metabolites. Futhermore, it presents emerging scientific data which suggest that, among well-known genistein and resveratrol, other phytoestrogens and their gut metabolites can act as epigenetic modulators with a possible impact on human health. The interconnection of dietary phytoestrogens' consumption with gut microbiota composition, epigenome and related preventive mechanisms is discussed. The current challenges and future perspectives in designing relevant research directions to explore the potential health benefits of dietary phytoestrogens are also explored.

Response to comment on "Genomic epidemiology of superspreading events in Austria reveals mutational dynamics and transmission properties of SARS-CoV-2".

Further analysis of SARS-CoV-2 genome sequencing data identifies several highly recurrent genetic variants with low allele frequencies, which, if filtered out, provide estimates consistent with tighter transmission bottlenecks.

Rapid, early and accurate SARS-CoV-2 detection using RT-qPCR in primary care: a prospective cohort study (REAP-1).

OBJECTIVES: We explore the importance of SARS-CoV-2 sentinel surveillance testing in primary care during a regional COVID-19 outbreak in Austria. DESIGN: Prospective cohort study. SETTING: A single sentinel practice serving 22 829 people in the ski-resort of Schladming-Dachstein. PARTICIPANTS: All 73 patients presenting with mild-to-moderate flu-like symptoms between 24 February and 03 April, 2020. INTERVENTION: Nasopharyngeal sampling to detect SARS-CoV-2 using real-time reverse transcriptase-quantitative PCR (RT-qPCR). OUTCOME MEASURES: We compared RT-qPCR at presentation with confirmed antibody status. We split the outbreak in two parts, by halving the period from the first to the last case, to characterise three cohorts of patients with confirmed infection: early acute (RT-qPCR reactive) in the first half; and late acute (reactive) and late convalescent (non-reactive) in the second half. For each cohort, we report the number of cases detected, the accuracy of RT-qPCR, the duration and variety of symptoms, and the number of viral clades present. RESULTS: Twenty-two patients were diagnosed with COVID-19 (eight early acute, seven late acute and seven late convalescent), 44 patients tested SARS-CoV-2 negative and 7 were excluded. The sensitivity of RT-qPCR was 100% among all acute cases, dropping to 68.1% when including convalescent. Test specificity was 100%. Mean duration of symptoms for each group were 2 days (range 1-4) among early acute, 4.4 days (1-7) among late acute and 8 days (2-12) among late convalescent. Confirmed infection was associated with loss of taste. Acute infection was associated with loss of taste, nausea/vomiting, breathlessness, sore throat and myalgia; but not anosmia, fever or cough. Transmission clusters of three viral clades (G, GR and L) were identified. CONCLUSIONS: RT-qPCR testing in primary care can rapidly and accurately detect SARS-CoV-2 among people with flu-like illness in a heterogeneous viral outbreak. Targeted testing in primary care can support national sentinel surveillance of COVID-19.

Characterization of CD8 T Cell-Mediated Mutations in the Immunodominant Epitope GP33-41 of Lymphocytic Choriomeningitis Virus.

Cytotoxic T lymphocytes (CTLs) represent key immune effectors of the host response against chronic viruses, due to their cytotoxic response to virus-infected cells. In response to this selection pressure, viruses may accumulate escape mutations that evade CTL-mediated control. To study the emergence of CTL escape mutations, we employed the murine chronic infection model of lymphocytic choriomeningitis virus (LCMV). We developed an amplicon-based next-generation sequencing pipeline to detect low frequency mutations in the viral genome and identified non-synonymous mutations in the immunodominant LCMV CTL epitope, GP33-41, in infected wildtype mice. Infected Rag2-deficient mice lacking CTLs did not contain such viral mutations. By using transgenic mice with T cell receptors specific to GP33-41, we characterized the emergence of viral mutations in this epitope under varying selection pressure. We investigated the two most abundant viral mutations by employing reverse genetically engineered viral mutants encoding the respective mutations. These experiments provided evidence that these mutations prevent activation and expansion of epitope-specific CD8 T cells. Our findings on the mutational dynamics of CTL escape mutations in a widely-studied viral infection model contributes to our understanding of how chronic viruses interact with their host and evade the immune response. This may guide the development of future treatments and vaccines against chronic infections.

The versatility of external quality assessment for the surveillance of laboratory and in vitro diagnostic performance: SARS-CoV-2 viral genome detection in Austria.

OBJECTIVES: External quality assessment (EQA) schemes provide information on individual and general analytical performance of participating laboratories and test systems. The aim of this study was to investigate the use and performance of SARS-CoV-2 virus genome detection systems in Austrian laboratories and their preparedness to face challenges associated with the pandemic. METHODS: Seven samples were selected to evaluate performance and estimate variability of reported results. Notably, a dilution series was included in the panel as a measure of reproducibility and sensitivity. Several performance criteria were evaluated for individual participants as well as in the cohort of all participants. RESULTS: A total of 109 laboratories participated and used 134 platforms, including 67 different combinations of extraction and PCR platforms and corresponding reagents. There were no false positives and 10 (1.2%) false negative results, including nine in the weakly positive sample (Ct ∼35.9, ∼640 copies/mL). Twenty (22%) laboratories reported results of mutation detection. Twenty-five (19%) test systems included amplification of human RNA as evidence of proper sampling. The overall linearity of Ct values from individual test systems for the dilution series was good, but inter-assay variability was high. Both operator-related and systematic failures appear to have caused incorrect results. CONCLUSIONS: Beyond providing certification for participating laboratories, EQA provides the opportunity for participants to evaluate their performance against others so that they may improve operating procedures and test systems. Well-selected EQA samples offer additional inferences to be made about assay sensitivity and reproducibility, which have practical applications.

Cutaneous Squamous Cell Carcinoma Development Is Associated with a Temporal Infiltration of ILC1 and NK Cells with Immune Dysfunctions.

NK cells and tissue-resident innate lymphoid cells (ILCs) are innate effectors found in the skin. To investigate their temporal dynamics and specific functions throughout the development of cutaneous squamous cell carcinoma (cSCC), we combined transcriptomic and immunophenotyping analyses in mouse and human cSCCs. We identified an infiltration of NK cells and ILC1s as well as the presence of a few ILC3s. Adoptive transfer of NK cells in NK cell‒ and ILC-deficient Nfil3-/- mice revealed a role for NK cells in early control of cSCC. During tumor progression, we identified a population skewing with the infiltration of atypical ILC1 secreting inflammatory cytokines but reduced levels of IFN-γ at the papilloma stage. NK cells and ILC1s were functionally impaired, with reduced cytotoxicity and IFN-γ secretion associated with the downregulation of activating receptors. They also showed a high degree of heterogeneity in mouse and human cSCCs with the expression of several markers of exhaustion, including TIGIT on NK cells and PD-1 and TIM-3 on ILC1s. Our data show an enrichment in inflammatory ILC1 at the precancerous stage together with impaired antitumor functions in NK cells and ILC1 that could contribute to the development of cSCC and thus suggest that future immunotherapies should take both ILC populations into account.

SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8+ T cell responses.

CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.

Severe Coronavirus Disease 2019 (COVID-19) is Associated With Elevated Serum Immunoglobulin (Ig) A and Antiphospholipid IgA Antibodies.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) frequently entails complications that bear similarities to autoimmune diseases. To date, there are little data on possible immunoglobulin (Ig) A-mediated autoimmune responses. Here, we aim to determine whether COVID-19 is associated with a vigorous total IgA response and whether IgA antibodies are associated with complications of severe illness. Since thrombotic events are frequent in severe COVID-19 and resemble hypercoagulation of antiphospholipid syndrome, our approach focused on antiphospholipid antibodies (aPL). METHODS: In this retrospective cohort study, clinical data and aPL from 64 patients with COVID-19 were compared from 3 independent tertiary hospitals (1 in Liechtenstein, 2 in Switzerland). Samples were collected from 9 April to 1 May 2020. RESULTS: Clinical records of 64 patients with COVID-19 were reviewed and divided into a cohort with mild illness (mCOVID; 41%), a discovery cohort with severe illness (sdCOVID; 22%) and a confirmation cohort with severe illness (scCOVID; 38%). Total IgA, IgG, and aPL were measured with clinical diagnostic kits. Severe illness was significantly associated with increased total IgA (sdCOVID, P = .01; scCOVID, P < .001), but not total IgG. Among aPL, both cohorts with severe illness significantly correlated with elevated anticardiolipin IgA (sdCOVID and scCOVID, P < .001), anticardiolipin IgM (sdCOVID, P = .003; scCOVID, P< .001), and anti-beta 2 glycoprotein-1 IgA (sdCOVID and scCOVID, P< .001). Systemic lupus erythematosus was excluded from all patients as a potential confounder. CONCLUSIONS: Higher total IgA and IgA-aPL were consistently associated with severe illness. These novel data strongly suggest that a vigorous antiviral IgA response, possibly triggered in the bronchial mucosa, induces systemic autoimmunity.

Genomic epidemiology of superspreading events in Austria reveals mutational dynamics and transmission properties of SARS-CoV-2.

Superspreading events shaped the coronavirus disease 2019 (COVID-19) pandemic, and their rapid identification and containment are essential for disease control. Here, we provide a national-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) superspreading during the first wave of infections in Austria, a country that played a major role in initial virus transmissions in Europe. Capitalizing on Austria's well-developed epidemiological surveillance system, we identified major SARS-CoV-2 clusters during the first wave of infections and performed deep whole-genome sequencing of more than 500 virus samples. Phylogenetic-epidemiological analysis enabled the reconstruction of superspreading events and charts a map of tourism-related viral spread originating from Austria in spring 2020. Moreover, we exploited epidemiologically well-defined clusters to quantify SARS-CoV-2 mutational dynamics, including the observation of low-frequency mutations that progressed to fixation within the infection chain. Time-resolved virus sequencing unveiled viral mutation dynamics within individuals with COVID-19, and epidemiologically validated infector-infectee pairs enabled us to determine an average transmission bottleneck size of 103 SARS-CoV-2 particles. In conclusion, this study illustrates the power of combining epidemiological analysis with deep viral genome sequencing to unravel the spread of SARS-CoV-2 and to gain fundamental insights into mutational dynamics and transmission properties.

Hepatocyte-intrinsic type I interferon signaling reprograms metabolism and reveals a novel compensatory mechanism of the tryptophan-kynurenine pathway in viral hepatitis.

The liver is a central regulator of metabolic homeostasis and serum metabolite levels. Hepatocytes are the functional units of the liver parenchyma and not only responsible for turnover of biomolecules but also act as central immune signaling platforms. Hepatotropic viruses infect liver tissue, resulting in inflammatory responses, tissue damage and hepatitis. Combining well-established in vitro and in vivo model systems with transcriptomic analyses, we show that type I interferon signaling initiates a robust antiviral immune response in hepatocytes. Strikingly, we also identify IFN-I as both, sufficient and necessary, to induce wide-spread metabolic reprogramming in hepatocytes. IFN-I specifically rewired tryptophan metabolism and induced hepatic tryptophan oxidation to kynurenine via Tdo2, correlating with altered concentrations of serum metabolites upon viral infection. Infected Tdo2-deficient animals displayed elevated serum levels of tryptophan and, unexpectedly, also vast increases in the downstream immune-suppressive metabolite kynurenine. Thus, Tdo2-deficiency did not result in altered serum homeostasis of the tryptophan to kynurenine ratio during infection, which seemed to be independent of hepatocyte-intrinsic compensation via the IDO-axis. These data highlight that inflammation-induced reprogramming of systemic tryptophan metabolism is tightly regulated in viral hepatitis.

Emergence of coronavirus disease 2019 (COVID-19) in Austria.

This is a report on the first identified cases of coronavirus disease 2019 (COVID-19) in Austria. The first documented case was a person who stayed in Kühtai, Tyrol, from 24 to 26 January 2020, and had been infected by a Chinese instructor in Starnberg (Germany) between 20 and 22 January. This counts as a German case since her diagnosis was eventually made in Munich (Germany) on 28 January. On 25 February, two cases imported from Italy were diagnosed in Innsbruck but again no secondary cases were identified in Austria. The first three infections of Austrian inhabitants were detected on 27 February in Vienna. The two resulting clusters finally included 6 (source of initial infection unknown) and 61 cases. Most likely, Italy was the source of the latter cluster. On 12 March the first fatal case of COVID-19 in Austria was reported, a 69-year-old Viennese who died in a Vienna hospital after returning from a cruise ship tour in Italy. On 6 March three autochthonously acquired cases were reported in the Tyrol, all related to the ski resort Ischgl. Of the first 14 Islandic COVID-19 cases infected in Ischgl, 11 had already returned to Iceland on 29 February. We consider that the incriminated barkeeper, who tested PCR positive on 7 March, was neither the primary case nor a superspreader. In our opinion, undetected transmission of SARS-CoV­2 had been ongoing in Ischgl prior to the first laboratory confirmed cases. Our data also underline that the introduction of SARS-CoV­2 into Austria was not one single event.

Tumor-Associated Neutrophils Dampen Adaptive Immunity and Promote Cutaneous Squamous Cell Carcinoma Development.

Cutaneous squamous cell carcinoma (cSCC) development has been linked to immune dysfunctions but the mechanisms are still unclear. Here, we report a progressive infiltration of tumor-associated neutrophils (TANs) in precancerous and established cSCC lesions from chemically induced skin carcinogenesis. Comparative in-depth gene expression analyses identified a predominant protumor gene expression signature of TANs in lesions compared to their respective surrounding skin. In addition, in vivo depletion of neutrophils delayed tumor growth and significantly increased the frequency of proliferating IFN-γ (interferon-γ)-producing CD8+ T cells. Mechanisms that limited antitumor responses involved high arginase activity, production of reactive oxygen species (ROS) and nitrite (NO), and the expression of programmed death-ligand 1 (PD-L1) on TAN, concomitantly with an induction of PD-1 on CD8+ T cells, which correlated with tumor size. Our data highlight the relevance of targeting neutrophils and PD-L1-PD-1 (programmed death-1) interaction in the treatment of cSCC.

Association between Internet Use Behavior and Palpitation among Adolescents: A Cross-Sectional Study of Middle School Children from Northwest Romania.

PURPOSE: The use of the internet is a tool and media literacy has become an essential skill among adolescents. Related to this behavior, some adolescents evoke cardiovascular effects. The purpose of this study was to explore a possible correlation between internet use behavior and occurrence of palpitations and related symptoms among a representative cohort of adolescents from the north-west region of Romania. METHOD: The study included students of seven middle schools from Northwest Romania. Participants completed an anonymous questionnaire consisting of 18 questions about internet use. RESULTS: In total, 1147 students responded to the study. Mean duration of daily internet usage was 2.57 h during school time and 3.57 h during the holidays. A total of 77% of adolescents had more than one symptom related to internet use, and 11% of them reported palpitations and related symptoms. We found an independent relation between palpitation and urban background, palpitations and the internet usage time interval 20:00-24:00, and palpitations and tobacco smoking. Strong heartbeats were independently associated with the time interval 12:00-16:00, tobacco smoking, and energy drink consumption. CONCLUSION: In our cohort, the most important factors associated with the occurrence of palpitations and related symptoms were the timeframe of internet usage and smoking.

Inverse Data-Driven Modeling and Multiomics Analysis Reveals Phgdh as a Metabolic Checkpoint of Macrophage Polarization and Proliferation.

Mechanistic or mammalian target of rapamycin complex 1 (mTORC1) is an important regulator of effector functions, proliferation, and cellular metabolism in macrophages. The biochemical processes that are controlled by mTORC1 are still being defined. Here, we demonstrate that integrative multiomics in conjunction with a data-driven inverse modeling approach, termed COVRECON, identifies a biochemical node that influences overall metabolic profiles and reactions of mTORC1-dependent macrophage metabolism. Using a combined approach of metabolomics, proteomics, mRNA expression analysis, and enzymatic activity measurements, we demonstrate that Tsc2, a negative regulator of mTORC1 signaling, critically influences the cellular activity of macrophages by regulating the enzyme phosphoglycerate dehydrogenase (Phgdh) in an mTORC1-dependent manner. More generally, while lipopolysaccharide (LPS)-stimulated macrophages repress Phgdh activity, IL-4-stimulated macrophages increase the activity of the enzyme required for the expression of key anti-inflammatory molecules and macrophage proliferation. Thus, we identify Phgdh as a metabolic checkpoint of M2 macrophages.

Type I Interferon Signaling Disrupts the Hepatic Urea Cycle and Alters Systemic Metabolism to Suppress T Cell Function.

Infections induce complex host responses linked to antiviral defense, inflammation, and tissue damage and repair. We hypothesized that the liver, as a central metabolic hub, may orchestrate systemic metabolic changes during infection. We infected mice with chronic lymphocytic choriomeningitis virus (LCMV), performed RNA sequencing and proteomics of liver tissue, and integrated these data with serum metabolomics at different infection phases. Widespread reprogramming of liver metabolism occurred early after infection, correlating with type I interferon (IFN-I) responses. Viral infection induced metabolic alterations of the liver that depended on the interferon alpha/beta receptor (IFNAR1). Hepatocyte-intrinsic IFNAR1 repressed the transcription of metabolic genes, including Otc and Ass1, which encode urea cycle enzymes. This led to decreased arginine and increased ornithine concentrations in the circulation, resulting in suppressed virus-specific CD8+ T cell responses and ameliorated liver pathology. These findings establish IFN-I-induced modulation of hepatic metabolism and the urea cycle as an endogenous mechanism of immunoregulation. VIDEO ABSTRACT.