High-Performance Genetically Encoded Green Fluorescent Biosensors for Intracellular l-Lactate.

l-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long generally been considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of l-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of l-lactate, we have developed a series of high-performance (ΔF/F = 15 to 30 in vitro), intensiometric, genetically encoded green fluorescent protein (GFP)-based intracellular l-lactate biosensors with a range of affinities. We evaluated these biosensors in cultured cells and demonstrated their application in an ex vivo preparation of Drosophila brain tissue. Using these biosensors, we were able to detect glycolytic oscillations, which we analyzed and mathematically modeled.

Health system barriers to the implementation of the national action plan to combat antimicrobial resistance in Vietnam: a scoping review.

BACKGROUND: Vietnam is among 11 countries in the Western Pacific region that has developed a National Action Plan for Antimicrobial Resistance (NAPCA). METHODS: This scoping review characterises health system barriers to the implementation of the Vietnam NAPCA, with reference to the WHO Health Systems Framework. RESULTS: Over 7 years, between 2013 and 2020, the Ministry of Health (MOH) of Vietnam has been implementing activities to achieve the six NAPCA objectives. They include revision of regulations needed for antimicrobial resistance (AMR) prevention programs; formation and operation of national management bodies; improvement of antimicrobial stewardship (AMS) in hospitals; maintenance of surveillance systems for AMR; provision of trainings on AMR and antibiotics use to doctors and pharmacists; and organization of nation-wide educational campaigns. Limited cooperation between MOH management bodies, shortages of human resource at all health system levels, a low degree of agreement between national and hospital guidelines on antibiotic use, low capability in the domestic supply of standardised drugs, and unequal training opportunities for lower-level health professionals present ongoing challenges. Actions suggested for the next period of the NAPCA include a final review of what has been achieved by the plan so far and evaluating the effectiveness of the different components of the plan. Different options on how to improve coordination across sectors in the development of a new NAPCA should be put forward. CONCLUSIONS: The 6-year implementation of the Vietnam NAPCA has yielded valuable lessons for AMS in Vietnam, guiding the development of future national plans, with a central focus on scaling up AMS in hospitals and promoting community AMS programs to combat AMR.

Enhanced Electron-Spin Coherence in a GaAs Quantum Emitter.

A spin-photon interface should operate with both coherent photons and a coherent spin to enable cluster-state generation and entanglement distribution. In high-quality devices, self-assembled GaAs quantum dots are near-perfect emitters of on-demand coherent photons. However, the spin rapidly decoheres via the magnetic noise arising from the host nuclei. Here, we address this drawback by implementing an all-optical nuclear-spin cooling scheme on a GaAs quantum dot. The electron-spin coherence time increases 156-fold from T_{2}^{*}=3.9 ns to 0.608 µs. The cooling scheme depends on a non-collinear term in the hyperfine interaction. The results show that such a term is present even though the strain is low and no external stress is applied. Our work highlights the potential of optically active GaAs quantum dots as fast, highly coherent spin-photon interfaces.

Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging.

L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of L-lactate are currently hampered by the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular L-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of L-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular L-lactate dynamics in mice.

Isomerization of bioactive acylhydrazones triggered by light or thiols.

The acylhydrazone unit is well represented in screening databases used to find ligands for biological targets, and numerous bioactive acylhydrazones have been reported. However, potential E/Z isomerization of the C=N bond in these compounds is rarely examined when bioactivity is assayed. Here we analysed two ortho-hydroxylated acylhydrazones discovered in a virtual drug screen for modulators of N-methyl-D-aspartate receptors and other bioactive hydroxylated acylhydrazones with structurally defined targets reported in the Protein Data Bank. We found that ionized forms of these compounds, which are populated under laboratory conditions, photoisomerize readily and the isomeric forms have markedly different bioactivity. Furthermore, we show that glutathione, a tripeptide involved with cellular redox balance, catalyses dynamic E⇄Z isomerization of acylhydrazones. The ratio of E to Z isomers in cells is determined by the relative stabilities of the isomers regardless of which isomer was applied. We conclude that E/Z isomerization may be a common feature of the bioactivity observed with acylhydrazones and should be routinely analysed.

Altered cleavage of human factor VIII at the B-domain and acidic region 3 interface enhances expression after gene therapy in hemophilia A mice.

BACKGROUND: Variants of human factor VIII (hFVIII) have been developed to further understand the structure and function of hFVIII and improve gene-based therapeutics. We have previously characterized several hFVIII variants of the furin cleavage site (1645-1648) with improved secretion. We have also identified a second cleavage site in the acidic region 3 (a3) (1657-1658) that becomes the primary hFVIII intracellular cleavage position in the absence of the furin site. We tested a hypothesis that modification of this site may confer additional functional advantages to hFVIII. OBJECTIVES: The aim of this study was to conduct the biochemical and functional characterization of hFVIII variants of the furin cleavage site, the a3 cleavage site, or in combination, both in vitro and in vivo after AAV mediated gene therapy. METHODS: Recombinant hFVIII variants of the furin cleavage site (hFVIII-Δ3), the a3 cleavage site (hFVIII-S1657P/D1658E [SP/DE]), or in combination (hFVIII-Δ3-SP/DE) were purified and characterized in vitro and in vivo. RESULTS: Recombinant hFVIII-Δ3, hFVIII-SP/DE, and hFVIII-Δ3-SP/DE variants all had comparable specific activity to B-domain deleted (BDD) hFVIII. Hemophilia A mice tolerant to hFVIII did not develop immune responses to hFVIII after protein challenge with these variants or after adeno-associated virus (AAV) delivery. Following AAV delivery, hFVIII-Δ3-SP/DE resulted in expression levels that were 2- to 5-fold higher than those with hFVIII-BDD in hemophilia A mice. CONCLUSION: The novel hFVIII-Δ3-SP/DE variant of the furin and a3 cleavage sites significantly improved secretion compared with hFVIII-BDD. This key feature of the Δ3-SP/DE variant provides a unique strategy that can be combined with other approaches to further improve factor VIII expression to achieve superior efficacy in AAV-based gene therapy for hemophilia A.

Utilization of Functionalized Metal-Organic Framework Nanoparticle as Targeted Drug Delivery System for Cancer Therapy.

Cancer is a multifaceted disease that results from the complex interaction between genetic and environmental factors. Cancer is a mortal disease with the biggest clinical, societal, and economic burden. Research on better methods of the detection, diagnosis, and treatment of cancer is crucial. Recent advancements in material science have led to the development of metal-organic frameworks, also known as MOFs. MOFs have recently been established as promising and adaptable delivery platforms and target vehicles for cancer therapy. These MOFs have been constructed in a fashion that offers them the capability of drug release that is stimuli-responsive. This feature has the potential to be exploited for cancer therapy that is externally led. This review presents an in-depth summary of the research that has been conducted to date in the field of MOF-based nanoplatforms for cancer therapeutics.

Application Prospects of MXenes Materials Modifications for Sensors.

The first two-dimensional (2D) substance sparked a boom in research since this type of material showed potential promise for applications in field sensors. A class of 2D transition metal nitrides, carbides, and carbonitrides are referred to as MXenes. Following the 2011 synthesis of Ti3C2 from Ti3AlC2, much research has been published. Since these materials have several advantages over conventional 2D materials, they have been extensively researched, synthesized, and studied by many research organizations. To give readers a general understanding of these well-liked materials, this review examines the structures of MXenes, discusses various synthesis procedures, and analyzes physicochemistry properties, particularly optical, electronic, structural, and mechanical properties. The focus of this review is the analysis of modern advancements in the development of MXene-based sensors, including electrochemical sensors, gas sensors, biosensors, optical sensors, and wearable sensors. Finally, the opportunities and challenges for further study on the creation of MXenes-based sensors are discussed.

Characterization of paramagnetic states in an organometallic nickel hydrogen evolution electrocatalyst.

Significant progress has been made in the bioinorganic modeling of the paramagnetic states believed to be involved in the hydrogen redox chemistry catalyzed by [NiFe] hydrogenase. However, the characterization and isolation of intermediates involved in mononuclear Ni electrocatalysts which are reported to operate through a NiI/III cycle have largely remained elusive. Herein, we report a NiII complex (NCHS2)Ni(OTf)2, where NCHS2 is 3,7-dithia-1(2,6)-pyridina-5(1,3)-benzenacyclooctaphane, that is an efficient electrocatalyst for the hydrogen evolution reaction (HER) with turnover frequencies of ~3,000 s-1 and a overpotential of 670 mV in the presence of trifluoroacetic acid. This electrocatalyst follows a hitherto unobserved HER mechanism involving C-H activation, which manifests as an inverse kinetic isotope effect for the overall hydrogen evolution reaction, and NiI/NiIII intermediates, which have been characterized by EPR spectroscopy. We further validate the possibility of the involvement of NiIII intermediates by the independent synthesis and characterization of organometallic NiIII complexes.

The endoplasmic reticulum kinase PERK interacts with the oxidoreductase ERO1 to metabolically adapt mitochondria.

Endoplasmic reticulum (ER) homeostasis requires molecular regulators that tailor mitochondrial bioenergetics to the needs of protein folding. For instance, calnexin maintains mitochondria metabolism and mitochondria-ER contacts (MERCs) through reactive oxygen species (ROS) from NADPH oxidase 4 (NOX4). However, induction of ER stress requires a quick molecular rewiring of mitochondria to adapt to new energy needs. This machinery is not characterized. We now show that the oxidoreductase ERO1⍺ covalently interacts with protein kinase RNA-like ER kinase (PERK) upon treatment with tunicamycin. The PERK-ERO1⍺ interaction requires the C-terminal active site of ERO1⍺ and cysteine 216 of PERK. Moreover, we show that the PERK-ERO1⍺ complex promotes oxidization of MERC proteins and controls mitochondrial dynamics. Using proteinaceous probes, we determined that these functions improve ER-mitochondria Ca2+ flux to maintain bioenergetics in both organelles, while limiting oxidative stress. Therefore, the PERK-ERO1⍺ complex is a key molecular machinery that allows quick metabolic adaptation to ER stress.

Isolation and Characterization of Heteroleptic Mononuclear Palladium(I) Complexes.

Catalytic transformations involving Pd(0)/Pd(II) catalytic cycles are very well known, and processes involving high-valent Pd(III) and Pd(IV) and low-valent Pd(I) intermediates have also gained interest in recent years. Although low-valent Pd(I) intermediates are proposed in these catalytic cycles, isolated and characterized mononuclear Pd(I) species are very rare. Herein, we report the isolation of two heteroleptic mononuclear Pd(I) complexes stabilized by dithiapyridinophane ligands that were fully characterized by single-crystal X-ray diffraction; EPR, IR, UV-vis spectroscopies; and computational studies. Excitingly, one of these Pd(I) complexes shows Kumada Csp3-Csp2 cross-coupling competency, and initial studies of the other shows direct evidence for Csp3-H bond activation proposed to occur at the Pd(I) center.

Changes in intestinal microbiota in postmenopausal oestrogen receptor-positive breast cancer patients treated with (neo)adjuvant chemotherapy.

This clinical study explored the associations between the intestinal microbiota, chemotherapy toxicity, and treatment response in postmenopausal oestrogen receptor positive breast cancer patients.Oestrogen receptor positive postmenopausal breast cancer patients were prospectively enroled in a multicentre cohort study and treated with 4 cycles of (neo)adjuvant adriamycin, cyclophosphamide (AC) followed by 4 cycles of docetaxel (D). Patients collected a faecal sample and completed a questionnaire before treatment, during AC, during D, and after completing AC-D. Chemotherapy toxicity and tumour response were determined. Intestinal microbiota was analysed by amplicon sequencing of the 16 S rRNA V4 gene-region. In total, 44 patients, including 18 neoadjuvant patients, were included, and 153 faecal samples were collected before AC-D (n = 44), during AC (n = 43), during D (n = 29), and after AC-D treatment (n = 37), 28 participants provided all four samples. In the whole group, observed species richness reduced during treatment (p = 0.042). The abundance of Proteobacteria, unclassified Enterobacterales, Lactobacillus, Ruminococcaceae NK4A214 group, Marvinbryantia, Christensenellaceae R7 group, and Ruminococcaceae UCG-005 changed significantly over time. Patients with any grade diarrhoea during docetaxel treatment had a significantly lower observed species richness compared to patients without diarrhoea. In the small group neoadjuvant treated patients, pathologic response was unrelated to baseline intestinal microbiota richness, diversity and composition. While the baseline microbiota was not predictive for pathologic response in a rather small group of neoadjuvant treated patients in our study, subsequent shifts in microbial richness, as well as the abundance of specific bacterial taxa, were observed during AC-D treatment in the whole group and the neoadjuvant group.

Fecal carriage of vanB antibiotic resistance gene affects adipose tissue function under vancomycin use.

Detrimental consequences of antibiotic treatment may include long-lasting disruption of the gut microbiota. Previous studies found no negative effects of antibiotics on metabolic health, although individualized responses were observed. Here, we aimed to investigate the subject-specific response to vancomycin use in tissue-specific insulin sensitivity by stratifying individuals based on the presence of antibiotic resistance genes (ARGs) or opportunistic pathogens (OPs) in the baseline fecal microbiota. Quantitative Polymerase Chain Reaction (qPCR) was used to detect ARGs and OPs in DNA isolated from fecal samples of 56 males with overweight/obesity (Body Mass Index: 25-35 kg/m2) and impaired glucose metabolism (fasting plasma glucose ≥5.6 mmol/L and/or 2-hour glucose 7.8-11.1 mmol/L). A two-step hyperinsulinemic-euglycemic clamp was performed to determine tissue-specific insulin sensitivity. Abdominal subcutaneous adipose tissue (AT) gene expression was assessed using Affymetrix microarray. Gut microbial composition was determined using the Human Intestinal Tract Chip (HITChip) microarray. At baseline, the vancomycin resistance gene vanB was present in 60% of our population. In individuals that were vanB-negative at baseline, AT insulin sensitivity (insulin-mediated suppression of plasma free fatty acids) improved during vancomycin use, while it decreased among vanB-positive individuals (% change post versus baseline: 14.1 ± 5.6 vs. -6.7 ± 7.5% (p = .042)). The vancomycin-induced increase in AT insulin sensitivity was accompanied by downregulation of inflammatory pathways and enrichment of extracellular matrix remodeling pathways in AT. In the vanB-positive group, well-known vanB-carrying bacteria, Enterococcus and Streptococcus, expanded in the gut microbiome. In conclusion, microbiome composition and adipose tissue biology were differentially affected by vancomycin treatment based on fecal vanB carriage.

Quantum interference of identical photons from remote GaAs quantum dots.

Photonic quantum technology provides a viable route to quantum communication1,2, quantum simulation3 and quantum information processing4. Recent progress has seen the realization of boson sampling using 20 single photons3 and quantum key distribution over hundreds of kilometres2. Scaling the complexity requires architectures containing multiple photon sources, photon counters and a large number of indistinguishable single photons. Semiconductor quantum dots are bright and fast sources of coherent single photons5-9. For applications, a roadblock is the poor quantum coherence on interfering single photons created by independent quantum dots10,11. Here we demonstrate two-photon interference with near-unity visibility (93.0 ± 0.8)% using photons from two completely separate GaAs quantum dots. The experiment retains all the emission into the zero phonon line-only the weak phonon sideband is rejected; temporal post-selection is not employed. By exploiting quantum interference, we demonstrate a photonic controlled-not circuit and an entanglement with fidelity of (85.0 ± 1.0)% between photons of different origins. The two-photon interference visibility is high enough that the entanglement fidelity is well above the classical threshold. The high mutual coherence of the photons stems from high-quality materials, diode structure and relatively large quantum dot size. Our results establish a platform-GaAs quantum dots-for creating coherent single photons in a scalable way.

The discourse of design: Patterns of TPACK Contribution during pre-service teacher learning design conversations.

Analysing pre-service teachers' learning design conversations in relation to Technological Pedagogical and Content Knowledge (TPACK) framework to understand their learning design practices has remained unexplored. This paper presents findings from a study of pre-service teachers' design discourses that identified how TPACK elements were used during their collaborative design of technology-enhanced lessons. Through thematic analysis of 81 design conversations in two cycles, it was found that pre-service teachers discussed design related issues, TPACK elements, and context in their design conversations with dominant references to design-related issues, substantial occurrences of single TPACK elements, and lower frequencies of integrated TPACK elements and context. Practical recommendations and a Design-TPACK or 'D-TPACK' framework were proposed to support pre-service teachers' learning design practices.

Electrocatalytic H2 evolution promoted by a bioinspired (N2S2)Ni(II) complex.

A bioinspired (N2S2)Ni(II) electrocatalyst is reported that produces H2 from CF3CO2H with a turnover frequency (TOF) of ∼1250 s-1 at low acid concentration (<0.043 M) in MeCN. A mechanism for the H2 production by this electrocatalyst is proposed and its activity is benchmarked against those of other reported molecular Ni H2 evolution electrocatalysts. The involvement of a hemilabile pyridyl group of the N2S2 ligand is proposed to mimic the role of a cysteine residue involved in the biological proton reduction performed by [NiFe] hydrogenases.

Preclinical assessment of an optimized AAV-FVIII vector in mice and non-human primates for the treatment of hemophilia A.

Extensive clinical data from liver-mediated gene therapy trials have shown that dose-dependent immune responses against the vector capsid may impair or even preclude transgene expression if not managed successfully with prompt immune suppression. The goal of this preclinical study was to generate an adeno-associated viral (AAV) vector capable of expressing therapeutic levels of B-domain deleted factor VIII (FVIII) at the lowest possible vector dose to minimize the potential Risk of a capsid-mediated immune response in the clinical setting. Here, we describe the studies that identified the investigational agent SPK-8011, currently being evaluated in a phase 1/2 study (NCT03003533) in individuals with hemophilia A. In particular, the potency of our second-generation expression cassettes was evaluated in mice and in non-human primates using two different bioengineered capsids (AAV-Spark100 and AAV-Spark200). At 2 weeks after gene transfer, primates transduced with 2 × 1012 vg/kg AAV-Spark100-FVIII or AAV-Spark200-FVIII expressed FVIII antigen levels of 13% ± 2% and 22% ± 6% of normal, respectively. Collectively, these preclinical results validate the feasibility of lowering the AAV capsid dose for a gene-based therapeutic approach for hemophilia A to a dose level orders of magnitude lower than the first-generation vectors in the clinic.

The Role of Intestinal Microbiota in Metastatic Colorectal Cancer Patients Treated With Capecitabine.

BACKGROUND: Previous pre-clinical research has indicated that the intestinal microbiota can potentiate anti-tumour efficacy of capecitabine and that capecitabine treatment impacts intestinal microbiota composition and diversity. Using a longitudinal design, this study explores the associations between the intestinal microbiota and treatment response in patients with metastatic colorectal cancer (mCRC) during capecitabine treatment. PATIENTS AND METHODS: Patients with mCRC treated with capecitabine were prospectively enrolled in a multicentre cohort study. Patients collected a faecal sample and completed a questionnaire before, during, and after three cycles of capecitabine. Several clinical characteristics, including tumour response, toxicity and antibiotic use were recorded. Intestinal microbiota were analysed by amplicon sequencing of the 16S rRNA V4 gene-region. RESULTS: Thirty-three patients were included. After three cycles of capecitabine, six patients (18%) achieved a partial response, 25 (76%) showed stable disease, and one (3%) experienced progressive disease. Of the 90 faecal samples were collected. Microbial diversity (α-diversity), community structure (ß-diversity), and bacterial abundance on phylum and genus level were not significantly different between responders and non-responders and were not significantly affected by three cycles of capecitabine. CONCLUSION: This is the first clinical study with longitudinal intestinal microbiota sampling in mCRC patients that explores the role of the intestinal microbiota during treatment with capecitabine. Intestinal microbiota composition and diversity before, during, and after three cycles of capecitabine were not associated with response in this study population. Capecitabine did not induce significant changes in the microbiota composition and diversity during the treatment period. Individual effects of antibiotics during capecitabine treatment were observed.

Intestinal Microbiota in Postmenopausal Breast Cancer Patients and Controls.

BACKGROUND: Previous preclinical and clinical research has investigated the role of intestinal microbiota in carcinogenesis. Growing evidence exists that intestinal microbiota can influence breast cancer carcinogenesis. However, the role of intestinal microbiota in breast cancer needs to be further investigated. This study aimed to identify the microbiota differences between postmenopausal breast cancer patients and controls. PATIENTS AND METHODS: This prospective cohort study compared the intestinal microbiota richness, diversity, and composition in postmenopausal histologically proven ER+/HER2- breast cancer patients and postmenopausal controls. Patients scheduled for (neo)adjuvant adriamycin, cyclophosphamide (AC), and docetaxel (D), or endocrine therapy (tamoxifen) were prospectively enrolled in a multicentre cohort study in the Netherlands. Patients collected a faecal sample and completed a questionnaire before starting systemic cancer treatment. Controls, enrolled from the National Dutch Breast Cancer Screening Programme, also collected a faecal sample and completed a questionnaire. Intestinal microbiota was analysed by amplicon sequencing of the 16S rRNA V4 gene region. RESULTS: In total, 81 postmenopausal ER+/HER2- breast cancer patients and 67 postmenopausal controls were included, resulting in 148 faecal samples. Observed species richness, Shannon index, and overall microbial community structure were not significantly different between breast cancer patients and controls. There was a significant difference in overall microbial community structure between breast cancer patients scheduled for adjuvant treatment, neoadjuvant treatment, and controls at the phylum (p = 0.042) and genus levels (p = 0.015). Dialister (p = 0.001) and its corresponding family Veillonellaceae (p = 0.001) were higher in patients scheduled for adjuvant treatment, compared to patients scheduled for neoadjuvant treatment. Additional sensitivity analysis to correct for the potential confounding effect of prophylactic antibiotic use, indicated no differences in microbial community structure between patients scheduled for neoadjuvant systemic treatment, adjuvant systemic treatment, and controls at the phylum (p = 0.471) and genus levels (p = 0.124). CONCLUSIONS: Intestinal microbiota richness, diversity, and composition are not different between postmenopausal breast cancer patients and controls. The increased relative abundance of Dialister and Veillonellaceae was observed in breast cancer patients scheduled for adjuvant treatment, which might be caused by a relative decrease in other bacteria due to prophylactic antibiotic administration rather than an absolute increase.

The magnetic resonance imaging of Leigh syndrome in a child.

ABSTRACT: Leigh syndrome is a rare progressive neurodegenerative disease with variable clinical presentations associated with mitochondrial dysfunction. However, the most common presentations are motor and intellectual developmental delays, with signs and symptoms of brainstem and basal ganglia involvement. We describe a 6-year-old boy with a history of delayed developmental milestones who presen-ted to our hospital due to unconscious status and respiratory distress syndrome. The patient underwent brain magnetic resonance imaging (MRI), and multiple subacute necrotic lesions were identified at the bilateral basal ganglia, thalamus, cerebral peduncles, brainstem, and cortical regions. DNA analysis was performed, which revealed muta-tions in SURF1. The patient experienced several relapses and died of respiratory failure and hospital-acquired infections, 3 years after the diagnosis of Leigh syndrome. Leigh syndrome should be considered in children with neurological problems and bilateral basal ganglia or brainstem abnormalities. Neurological MRI can be useful for guiding clinicians in ordering the most appropriate enzymatic and genetic analyses for further diagnosis.