Model compounds for evaluating the reactivity of amphetamine-type stimulants.

The use of controlled precursors for reaction optimisation is not always practical. One approach to limiting the use of controlled substances is to instead use 'model compounds'. Herein, two model compounds resembling norephedrine and ephedrine were selected based on their (i) structural similarity (i.e., presence of key functional groups) and (ii) availability from multiple suppliers without restriction. Model compounds 2-amino-1-phenylethanol and 2-(methylamino)-1-phenylethanol (halostachine), were compared to norephedrine and pseudoephedrine by firstly subjecting them to transformations known in the synthesis of amphetamines, and secondly, comparing the compounds using colourimetric spot tests, FTIR and NMR.

A transparent approach: Openness in forensic science reporting.

There have been numerous calls for increased transparency and disclosure in forensic science. However, there is a paucity of guidance on how to achieve this transparency in reports, and the impacts it may have on criminal justice proceedings. We describe one multi-disciplinary forensic laboratory's journey to fully transparent reporting, disclosing matters of scientific relevance and importance. All expert reports across 17 disciplines now contain information regarding the fundamental principles and methodology, validity and error, assumptions and limitations, competency testing and quality assurance, cognitive factors, and areas of scientific controversy. Staff support for transparent reporting increased following introduction, with most reporting largely positive impacts. A slight increase in questioning in court has been experienced, with increased legal attention paid to the indicia of scientific validity. Transparency in expert forensic science reports is possible, and can improve the use of scientific evidence in courts without compromising the timeliness of service.

Tlr9 deficiency in B cells leads to obesity by promoting inflammation and gut dysbiosis.

Toll-like receptor 9 (TLR9) recognizes bacterial, viral and self DNA and play an important role in immunity and inflammation. However, the role of TLR9 in obesity is less well-studied. Here, we generate B-cell-specific Tlr9-deficient (Tlr9fl/fl/Cd19Cre+/-, KO) B6 mice and model obesity using a high-fat diet. Compared with control mice, B-cell-specific-Tlr9-deficient mice exhibited increased fat tissue inflammation, weight gain, and impaired glucose and insulin tolerance. Furthermore, the frequencies of IL-10-producing-B cells and marginal zone B cells were reduced, and those of follicular and germinal center B cells were increased. This was associated with increased frequencies of IFNγ-producing-T cells and increased follicular helper cells. In addition, gut microbiota from the KO mice induced a pro-inflammatory state leading to immunological and metabolic dysregulation when transferred to germ-free mice. Using 16 S rRNA gene sequencing, we identify altered gut microbial communities including reduced Lachnospiraceae, which may play a role in altered metabolism in KO mice. We identify an important network involving Tlr9, Irf4 and Il-10 interconnecting metabolic homeostasis, with the function of B and T cells, and gut microbiota in obesity.

TLR5-deficiency controls dendritic cell subset development in an autoimmune diabetes-susceptible model.

Introduction: The incidence of the autoimmune disease, type 1 diabetes (T1D), has been increasing worldwide and recent studies have shown that the gut microbiota are associated with modulating susceptibility to T1D. Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and is widely expressed on many cells, including dendritic cells (DCs), which are potent antigen-presenting cells (APCs). TLR5 modulates susceptibility to obesity and alters metabolism through gut microbiota; however, little is known about the role TLR5 plays in autoimmunity, especially in T1D. Methods: To fill this knowledge gap, we generated a TLR5-deficient non-obese diabetic (NOD) mouse, an animal model of human T1D, for study. Results: We found that TLR5-deficiency led to a reduction in CD11c+ DC development in utero, prior to microbial colonization, which was maintained into adulthood. This was associated with a bias in the DC populations expressing CD103, with or without CD8α co-expression, and hyper-secretion of different cytokines, both in vitro (after stimulation) and directly ex vivo. We also found that TLR5-deficient DCs were able to promote polyclonal and islet antigen-specific CD4+ T cell proliferation and proinflammatory cytokine secretion. Interestingly, only older TLR5-deficient NOD mice had a greater risk of developing spontaneous T1D compared to wild-type mice. Discussion: In summary, our data show that TLR5 modulates DC development and enhances cytokine secretion and diabetogenic CD4+ T cell responses. Further investigation into the role of TLR5 in DC development and autoimmune diabetes may give additional insights into the pathogenesis of Type 1 diabetes.

Amide-to-chloroalkene substitution for overcoming intramolecular acyl transfer challenges in hexapeptidic neuromedin U receptor 2 agonists.

CPN-116 is a peptidic agonist that activates human neuromedin U receptor type 2 (NMUR2) but suffers from chemical instability due to inherent backbone isomerization on the Dap residue. To address this, a Leu-Dap-type (Z)-chloroalkene dipeptide isostere was synthesized diastereoselectively as a surrogate of the Leu-Dap peptide bond to develop a (Z)-chloroalkene analogue of CPN-116. The synthesized CPN-116 analogue is stable in 1.0 M phosphate buffer (pH 7.4) without backbone isomerization and can activate NMUR2 with similar potency to CPN-116 at nM concentrations (EC50 = 1.0 nM).

Photocatalytic O- to S-Rearrangement of Tertiary Cyclopropanols.

Despite the importance of heteroatom-substituted cyclopropane derivatives in drug design and organic synthesis, cyclopropanethiols remain critically underexplored. Inspired by the wide use of the Newman-Kwart rearrangement to access valuable thiophenols from phenol feedstocks, we report the development of a photocatalytic approach for efficient ambient temperature aliphatic O- to S-rearrangement on tertiary cyclopropanol derivatives. After demonstrating that a range of cyclopropanethiols-that are difficult to access by other methods-can be obtained with this strategy, we show that these rearranged products can be easily hydrolyzed and further derivatized. We conclude this study with mechanistic findings that enabled an initial extension of this approach toward other classes of aliphatic alcohols.

Sustained inflation improves initial lung aeration in newborn rabbits with a diaphragmatic hernia.

BACKGROUND: Infants with a congenital diaphragmatic hernia (DH) have underdeveloped lungs and require mechanical ventilation after birth, but the optimal approach is unknown. We hypothesised that sustained inflation (SI) increases lung aeration in newborn kittens with a DH. METHODS: In pregnant New Zealand white rabbits, a left-sided DH was induced in two fetal kittens per doe at 24-days gestation (term = 32 days); litter mates acted as controls. DH and control kittens were delivered by caesarean section at 30 days, intubated and mechanically ventilated (7-10 min) with either an SI followed by intermittent positive pressure ventilation (IPPV) or IPPV throughout. The rate and uniformity of lung aeration was measured using phase-contrast X-ray imaging. RESULTS: Lung weights in DH kittens were ~57% of controls. An SI increased the rate and uniformity of lung aeration in DH kittens, compared to IPPV, and increased dynamic lung compliance in both control and DH kittens. However, this effect of the SI was lost when ventilation changed to IPPV. CONCLUSION: While an SI improved the rate and uniformity of lung aeration in both DH and control kittens, greater consideration of the post-SI ventilation strategy is required to sustain this benefit. IMPACT: Compared to intermittent positive pressure ventilation (IPPV), an initial sustained inflation (SI) increased the rate and uniformity of lung aeration after birth. However, this initial benefit is rapidly lost following the switch to IPPV. The optimal approach for ventilating CDH infants at birth is unknown. While an SI improves lung aeration in immature lungs, its effect on the hypoplastic lung is unknown. This study has shown that an SI greatly improves lung aeration in the hypoplastic lung. This study will guide future studies examining whether an SI can improve lung aeration in infants with a CDH.

Facial fanning reduces heart rate but not tolerance to a simulated hemorrhagic challenge following exercise heat stress in young healthy humans.

We investigated whether reducing face skin temperature alters arterial blood pressure control and lower body negative pressure (LBNP) tolerance after exercise heat stress. Eight subjects (1 female; age, 27 ± 9 yr) exercised at ∼63% V̇o2max until core temperature had increased ∼1.5°C before undergoing LBNP to presyncope either with fanning to return face skin temperature to baseline (Δ-5°C, Fan trial) or without (No Fan trial). LBNP tolerance was quantified as cumulative stress index (CSI; mmHg·min). Before LBNP, whole body and face skin temperatures were elevated from baseline in both trials (38.0 ± 0.5°C and 36.3 ± 0.5°C, respectively, both P < 0.001). During LBNP, face skin temperature decreased in the Fan trial (30.9 ± 1.0°C) but was unchanged in the No Fan trial (36.1 ± 0.6°C, between trials P < 0.001). Mean arterial pressure was not different between trials (P = 0.237) and was similarly reduced at presyncope in both trials (from 82 ± 7 to 67 ± 8 mmHg, P < 0.001). During LBNP, heart rate was attenuated in the Fan trial at Mid LBNP (146 ± 16 vs. 158 ± 12 beats/min, P = 0.036) and at peak heart rate (158 ± 15 vs. 170 ± 15 beats/min; P < 0.001). LBNP tolerance was not different between trials (321 ± 248 vs. 328 ± 115 mmHg·min, P = 0.851). In exercise heat-stressed individuals, lowering face skin temperature to normothermic values suppressed heart rate thereby altering cardiovascular control during a simulated hemorrhagic challenge without reducing tolerance.

NLRP6 deficiency expands a novel CD103+ B cell population that confers immune tolerance in NOD mice.

Introduction: Gut microbiota have been linked to modulating susceptibility to Type 1 diabetes; however, there are many ways in which the microbiota interact with host cells, including through microbial ligand binding to intracellular inflammasomes (large multi-subunit proteins) to initiate immune responses. NLRP6, a microbe-recognizing inflammasome protein, is highly expressed by intestinal epithelial cells and can alter susceptibility to cancer, obesity and Crohn's disease; however, the role of NLRP6 in modulating susceptibility to autoimmune diabetes, was previously unknown. Methods: We generated NLRP6-deficient Non-obese diabetic (NOD) mice to study the effect of NLRP6-deficiency on the immune cells and susceptibility to Type 1 diabetes development. Results: NLRP6-deficient mice exhibited an expansion of CD103+ B cells and were protected from type 1 diabetes. Moreover, NLRP6-deficient CD103+ B cells express regulatory markers, secreted higher concentrations of IL-10 and TGFb1 cytokines and suppressed diabetogenic T cell proliferation, compared to NLRP6-sufficient CD103+ B cells. Microarray analysis of NLRP6-sufficient and -deficient CD103+ B cells identified 79 significantly different genes including genes regulated by lipopolysaccharide (LPS), tretinoin, IL-10 and TGFb, which was confirmed in vitro following LPS stimulation. Furthermore, microbiota from NLRP6-deficient mice induced CD103+ B cells in colonized NLRP6-sufficient germ-free mice; however, the long-term maintenance of the CD103+ B cells required the absence of NLRP6 in the hosts, or continued exposure to microbiota from NLRP6-deficient mice. Discussion: Together, our data indicate that NLRP6 deficiency promotes expansion and maintenance of a novel TGF -dependent CD103+ Breg population. Thus, targeting NLRP6 therapeutically may prove clinically useful.

Effect of prenatal diaphragmatic hernia on pulmonary arterial morphology.

Congenital diaphragmatic hernia (CDH) is a major cause of severe lung hypoplasia and pulmonary hypertension in the newborn. While the pulmonary hypertension is thought to result from abnormal vascular development and arterial vasoreactivity, the anatomical changes in vascular development are unclear. We have examined the 3D structure of the pulmonary arterial tree in rabbits with a surgically induced diaphragmatic hernia (DH). Fetal rabbits (n = 6) had a left-sided DH created at gestational day 23 (GD23), delivered at GD30, and briefly ventilated; sham-operated litter mates (n = 5) acted as controls. At postmortem the pulmonary arteries were filled with a radio-opaque resin before the lungs were scanned using computed tomography (CT). The 3D reconstructed images were analyzed based on vascular branching hierarchy using the software Avizo 2020.2. DH significantly reduced median number of arteries (2,579 (8440) versus 576 (442), p = .017), artery numbers per arterial generation, mean total arterial volume (43.5 ± 8.4 vs. 19.9 ± 3.1 µl, p = .020) and mean total arterial cross-sectional area (82.5 ± 2.3 vs. 28.2 ± 6.2 mm2 , p =.036). Mean arterial radius was increased in DH kittens between the eighth and sixth branching generation and mean arterial length between the sixth and 28th branching generation. A DH in kittens resulted in threefold reduction in pulmonary arterial cross-sectional area, primarily due to reduced arterial branching. Thus, the reduction in arterial cross-sectional area could be a major contributor to pulmonary hypertension infants with CDH.

Set7 methyltransferase roles in myocardial protection from chronic stressors.

Epigenome changes in chronic states of cardiovascular stress including diabetes, pressure overload and cardiomyopathies frequently involve changes in open chromatin and post-translation modifications of histone lysine residues at specific amino acid positions by acetylation, methylation and phosphorylation. Since the discovery of Set7 as an important regulator of histone H3 lysine 4 methylation state, there has been wide interest in its role in cardiovascular remodeling and cardiac dysfunction. Recent transcriptome and Fourier transform infrared spectroscopy analyses and in vivo assessments of cardiac function by Lunardon and colleagues now reveal a clear role of Set7 in the regulation of the extracellular matrix composition and cardiac hypertrophy in response to chronic isoproterenol induced cardiac stress.

Gene expression profiling in NOD mice reveals that B cells are highly educated by the pancreatic environment during autoimmune diabetes.

AIMS/HYPOTHESIS: B cells play an important role in driving the development of type 1 diabetes; however, it remains unclear how they contribute to local beta cell destruction during disease progression. Here, we use gene expression profiling of B cell subsets identified in inflamed pancreatic tissue to explore their primary functional role during the progression of autoimmune diabetes. METHODS: Transcriptional profiling was performed on FACS-sorted B cell subsets isolated from pancreatic islets and the pancreatic lymph nodes of NOD mice. RESULTS: B cells are highly modified by the inflamed pancreatic tissue and can be distinguished by their transcriptional profile from those in the lymph nodes. We identified both a discrete and a core shared gene expression profile in islet CD19+CD138- and CD19+CD138+ B cell subsets, the latter of which is known to have enriched autoreactivity during diabetes development. On localisation to pancreatic islets, compared with CD138- B cells, CD138+ B cells overexpress genes associated with adhesion molecules and growth factors. Their shared signature consists of gene expression changes related to the differentiation of antibody-secreting cells and gene regulatory networks associated with IFN signalling pathways, proinflammatory cytokines and Toll-like receptor (TLR) activation. Finally, abundant TLR7 expression was detected in islet B cells and was enhanced specifically in CD138+ B cells. CONCLUSIONS/INTERPRETATION: Our study provides a detailed transcriptional analysis of islet B cells. Specific gene signatures and interaction networks have been identified that point towards a functional role for B cells in driving autoimmune diabetes.

Transporter-dependent uptake and metabolism of myocardial interstitial serotonin in the rat heart.

To investigate the roles of the serotonin (5-HT) transporter (SERT) and plasma membrane monoamine transporter (PMAT) in 5-HT uptake and its metabolism in the heart, we monitored myocardial interstitial levels of 5-HT and 5-HIAA, a metabolite of 5-HT by monoamine oxidase (MAO), in anesthetized rats using a microdialysis technique. Fluoxetine (SERT inhibitor), decynium-22 (PMAT inhibitor), or their mixture was locally administered by reverse-microdialysis for 60 min. Subsequently, pargyline (MAO inhibitor) was co-administered. Fluoxetine rapidly increased dialysate 5-HT concentration, while decynium-22 gradually increased it. The mixture induced a larger increase in dialysate 5-HT concentration compared to fluoxetine or decynium-22 alone. Fluoxetine increased dialysate 5-HIAA concentration, and this increase was abolished by pargyline. Decynium-22 and the mixture did not change dialysate 5-HIAA concentration, which were not affected by pargyline. Both SERT and PMAT regulate myocardial interstitial 5-HT levels by its uptake; however, 5-HT uptake via PMAT leads to 5-HT metabolism by MAO.

Circadian rhythms and pancreas physiology: A review.

Type 2 diabetes mellitus, obesity and metabolic syndrome are becoming more prevalent worldwide and will present an increasingly challenging burden on healthcare systems. These interlinked metabolic abnormalities predispose affected individuals to a plethora of complications and comorbidities. Furthermore, diabetes is estimated by the World Health Organization to have caused 1.5 million deaths in 2019, with this figure projected to rise in coming years. This highlights the need for further research into the management of metabolic diseases and their complications. Studies on circadian rhythms, referring to physiological and behavioral changes which repeat approximately every 24 hours, may provide important insight into managing metabolic disease. Epidemiological studies show that populations who are at risk of circadian disruption such as night shift workers and regular long-haul flyers are also at an elevated risk of metabolic abnormalities such as insulin resistance and obesity. Aberrant expression of circadian genes appears to contribute to the dysregulation of metabolic functions such as insulin secretion, glucose homeostasis and energy expenditure. The potential clinical implications of these findings have been highlighted in animal studies and pilot studies in humans giving rise to the development of circadian interventions strategies including chronotherapy (time-specific therapy), time-restricted feeding, and circadian molecule stabilizers/analogues. Research into these areas will provide insights into the future of circadian medicine in metabolic diseases. In this review, we discuss the physiology of metabolism and the role of circadian timing in regulating these metabolic functions. Also, we review the clinical aspects of circadian physiology and the impact that ongoing and future research may have on the management of metabolic disease.

Increased contribution of KCa channels to muscle contraction induced vascular and blood flow responses in sedentary and exercise trained ZFDM rats.

In resistance arteries, endothelium-dependent hyperpolarization (EDH)-mediated vasodilatation is depressed in diabetes. We hypothesized that downregulation of KCa channel derived EDH reduces exercise-induced vasodilatation and blood flow redistribution in diabetes. To test this hypothesis, we evaluated vascular function in response to hindlimb muscle contraction, and the contribution of KCa channels in anaesthetised ZFDM, metabolic disease rats with type 2 diabetes. We also tested whether exercise training ameliorated the vascular response. Using in vivo microangiography, the hindlimb vasculature was visualized before and after rhythmic muscle contraction (0.5 s tetanus every 3 s, 20 times) evoked by sciatic nerve stimulation (40 Hz). Femoral blood flow of the contracting hindlimb was simultaneously measured by an ultrasonic flowmeter. The contribution of KCa channels was investigated in the presence and absence of apamin and charybdotoxin. We found that vascular and blood flow responses to muscle contraction were significantly impaired at the level of small artery segments in ZFDM fa/fa rats compared to its lean control fa/+ rats. The contribution of KCa channels was also smaller in fa/fa than in fa/+ rats. Low-intensity exercise training for 12 weeks in fa/fa rats demonstrated minor changes in the vascular and blood flow response to muscle contraction. However, the KCa-derived component in the response to muscle contraction was much greater in exercise trained than in sedentary fa/fa rats. These data suggest that exercise training increases the contribution of KCa channels among endothelium-dependent vasodilatory mechanisms to maintain vascular and blood flow responses to muscle contraction in this metabolic disease rat model. KEY POINTS: Microvascular dysfunction in type 2 diabetes impairs blood flow redistribution during exercise and limits the performance of skeletal muscle and may cause early fatigability. Endothelium-dependent hyperpolarization (EDH), which mediates vasodilatation in resistance arteries, is known to be depressed in animals with diabetes. Here, we report that low-intensity exercise training in ZFDM rats increased the KCa channel-derived component in the vasodilator responses to muscle contraction compared to that in sedentary rats, partly as a result of the increase in KCNN3 expression. These results suggest that low-intensity exercise training improves blood flow redistribution in contracting skeletal muscle in metabolic disease with diabetes via upregulation of EDH.

Endothelial Natriuretic Peptide Receptor 1 Play Crucial Role for Acute and Chronic Blood Pressure Regulation by Atrial Natriuretic Peptide.

BACKGROUND: ANP (atrial natriuretic peptide), acting through NPR1 (natriuretic peptide receptor 1), provokes hypotension. Such hypotension is thought to be due to ANP inducing vasodilation via NPR1 in the vasculature; however, the underlying mechanism remains unclear. Here, we investigated the mechanisms of acute and chronic blood pressure regulation by ANP. METHODS AND RESULTS: Immunohistochemical analysis of rat tissues revealed that NPR1 was abundantly expressed in endothelial cells and smooth muscle cells of small arteries and arterioles. Intravenous infusion of ANP significantly lowered systolic blood pressure in wild-type mice. ANP also significantly lowered systolic blood pressure in smooth muscle cell-specific Npr1-knockout mice but not in endothelial cell-specific Npr1-knockout mice. Moreover, ANP significantly lowered systolic blood pressure in Nos3-knockout mice. In human umbilical vein endothelial cells, treatment with ANP did not influence nitric oxide production or intracellular Ca2+ concentration, but it did hyperpolarize the cells. ANP-induced hyperpolarization of human umbilical vein endothelial cells was inhibited by several potassium channel blockers and was also abolished under knockdown of RGS2 (regulator of G-protein signaling 2), an GTPase activating protein in G-protein α-subunit. ANP increased Rgs2 mRNA expression in human umbilical vein endothelial cells but failed to lower systolic blood pressure in Rgs2-knockout mice. Endothelial cell-specific Npr1-overexpressing mice exhibited lower blood pressure than did wild-type mice independent of RGS2, and showed dilation of arterial vessels on synchrotron radiation microangiography. CONCLUSIONS: Together, these results indicate that vascular endothelial NPR1 plays a crucial role in ANP-mediated blood pressure regulation, presumably by a mechanism that is RGS2-dependent in the acute phase and RGS2-independent in the chronic phase.

IgM-associated gut bacteria in obesity and type 2 diabetes in C57BL/6 mice and humans.

AIMS/HYPOTHESIS: IgM is the primary antibody produced by B cells and we hypothesise that IgM antibodies to gut microbiota may play a role in immunometabolism in obesity and type 2 diabetes. To test our hypothesis, we used B6 mice deficient in activation-induced cytidine deaminase (Aid-/- [also known as Aicda-/-]) which secrete only IgM antibodies, and human faecal samples. METHODS: We studied the immunometabolic effects and gut microbial changes in high-fat-diet-induced obesity (HFDIO) in Aid-/- B6 mice compared with wild-type mice. To determine similarities between mice and humans, human stool samples were collected from children and adolescents who were obese with normal glucose tolerance (NGT), obese with glucose intolerance (IGT), or obese and newly diagnosed with type 2 diabetes, for faecal microbiota transplant (FMT) into germ-free (GF) B6 mice and we assessed IgM-bound bacteria and immune responses. RESULTS: Compared with wild-type mice, Aid-/- B6 mice developed exacerbated HFDIO due to abundant levels of IgM. FMT from Aid-/- B6 to GF B6 mice promoted greater weight gain in recipient mice compared with FMT using wild-type mouse faecal microbiota. Obese youth with type 2 diabetes had more IgM-bound gut bacteria. Using the stools from the obese youth with type 2 diabetes for FMT to GF B6 mice, we observed that the gut microbiota promoted body weight gain and impaired glucose tolerance in the recipient GF B6 mice. Importantly, some clinical features of these obese young individuals were mirrored in the GF B6 mice following FMT. CONCLUSIONS/INTERPRETATION: Our results suggest that IgM-bound gut microbiota may play an important role in the immuno-pathogenesis of obesity and type 2 diabetes, and provide a novel link between IgM in obesity and type 2 diabetes in both mice and humans. DATA AVAILABILITY: The 16s rRNA sequencing datasets supporting the current study have been deposited in the NCBI SRA public repository ( https://www.ncbi.nlm.nih.gov/sra ; accession no. SAMN18796639).