An in vitro neurogenetics platform for precision disease modeling in the mouse.

The power and scope of disease modeling can be markedly enhanced through the incorporation of broad genetic diversity. The introduction of pathogenic mutations into a single inbred mouse strain sometimes fails to mimic human disease. We describe a cross-species precision disease modeling platform that exploits mouse genetic diversity to bridge cell-based modeling with whole organism analysis. We developed a universal protocol that permitted robust and reproducible neural differentiation of genetically diverse human and mouse pluripotent stem cell lines and then carried out a proof-of-concept study of the neurodevelopmental gene DYRK1A. Results in vitro reliably predicted the effects of genetic background on Dyrk1a loss-of-function phenotypes in vivo. Transcriptomic comparison of responsive and unresponsive strains identified molecular pathways conferring sensitivity or resilience to Dyrk1a1A loss and highlighted differential messenger RNA isoform usage as an important determinant of response. This cross-species strategy provides a powerful tool in the functional analysis of candidate disease variants identified through human genetic studies.

Tmod2 is a regulator of cocaine responses through control of striatal and cortical excitability, and drug-induced plasticity.

Drugs of abuse induce neuroadaptations, including synaptic plasticity, that are critical for transition to addiction, and genes and pathways that regulate these neuroadaptations are potential therapeutic targets. Tropomodulin 2 (Tmod2) is an actin-regulating gene that plays an important role in synapse maturation and dendritic arborization and has been implicated in substance-abuse and intellectual disability in humans. Here we mine the KOMP2 data and find that Tmod2 knockout mice show emotionality phenotypes that are predictive of addiction vulnerability. Detailed addiction phenotyping showed that Tmod2 deletion does not affect the acute locomotor response to cocaine administration. However, sensitized locomotor responses are highly attenuated in these knockouts, indicating perturbed drug-induced plasticity. In addition, Tmod2 mutant animals do not self-administer cocaine indicating lack of hedonic responses to cocaine. Whole brain MR imaging shows differences in brain volume across multiple regions although transcriptomic experiments did not reveal perturbations in gene co-expression networks. Detailed electrophysiological characterization of Tmod2 KO neurons, showed increased spontaneous firing rate of early postnatal and adult cortical and striatal neurons. Cocaine-induced synaptic plasticity that is critical for sensitization is either missing or reciprocal in Tmod2 KO nucleus accumbens shell medium spiny neurons, providing a mechanistic explanation of the cocaine response phenotypes. Combined, these data collected from both males and females, provide compelling evidence that Tmod2 is a major regulator of plasticity in the mesolimbic system and regulates the reinforcing and addictive properties of cocaine.Significance statement We identify, characterize, and establish tropomodulin 2 (Tmod2), an actin-regulating gene exclusively expressed in neurons, as an important regulator of addiction-related phenotypes. We show that Tmod2, knockout mice (Tmod2 KO) exhibit phenotypes that are predictive of addiction. In detailed addiction phenotyping, we find the Tmod2 regulates cocaine sensitization and self-administration. We explore anatomical, transcriptional, electrophysiological mechanisms of this regulation. Combined these studies provide compelling evidence that Tmod2 is critical for synaptic plasticity necessary for transition to addiction.

Gastrointestinal manifestations of critical ill heatstroke patients and their associations with outcomes: A multicentre, retrospective, observational study.

BACKGROUND: Extreme heat exposure is a growing health problem, and the effects of heat on the gastrointestinal (GI) tract is unknown. This study aimed to assess the incidence of GI symptoms associated with heatstroke and its impact on outcomes. AIM: To assess the incidence of GI symptoms associated with heatstroke and its impact on outcomes. METHODS: Patients admitted to the intensive care unit (ICU) due to heatstroke were included from 83 centres. Patient history, laboratory results, and clinically relevant outcomes were recorded at ICU admission and daily until up to day 15, ICU discharge, or death. GI symptoms, including nausea/vomiting, diarrhoea, flatulence, and bloody stools, were recorded. The characteristics of patients with heatstroke concomitant with GI symptoms were described. Multivariable regression analyses were performed to determine significant predictors of GI symptoms. RESULTS: A total of 713 patients were included in the final analysis, of whom 132 (18.5%) patients had at least one GI symptom during their ICU stay, while 26 (3.6%) suffered from more than one symptom. Patients with GI symptoms had a significantly higher ICU stay compared with those without. The mortality of patients who had two or more GI symptoms simultaneously was significantly higher than that in those with one GI symptom. Multivariable logistic regression analysis revealed that older patients with a lower GCS score on admission were more likely to experience GI symptoms. CONCLUSION: The GI manifestations of heatstroke are common and appear to impact clinically relevant hospitalization outcomes.

Antibodies against SARS-CoV-2 non-structural protein 3 cross-react with human muscle cells and neuroglial cells.

Coronavirus Disease 2019 (COVID-19) vaccines protect the public and limit viral spread. However, inactivated viral vaccines use the whole virus particle, which contains many non-capsid proteins that may cause adverse immune responses. A report has found that the ADP-ribose-binding domains of SARS-CoV-2 non-structural protein 3 (NSP3) and human poly(ADP-ribose) polymerase family member 14 (PARP14) share a significant degree of homology. Here, we further show that antibodies against 2019 novel SARS-like coronavirus (SARS-CoV-2) NSP3 can bind human PARP14 protein. However, when G159R + G162R mutations were introduced into NSP3, the antibody titer against human PARP14 decreased 14-fold. Antibodies against SARS-CoV-2 NSP3 can cross-react with human skeletal muscle cells and astrocytes, but not human embryonic kidney 293T cells. However, when G159R + G162R mutations were introduced into NSP3, the cross-reaction was largely inhibited. The results imply that COVID-19 patients with high antibody titers against NSP3 may have high risks of muscular and/or neurological complications. And the possible strategies to improve the safety of inactivated viral vaccines are also discussed.

Singlet oxygen induces cell wall thickening and stomatal density reducing by transcriptome reprogramming.

Singlet oxygen (1O2) has a very short half-life of 10-5 s; however, it is a strong oxidant that causes growth arrest and necrotic lesions on plants. Its signaling pathway remains largely unknown. The Arabidopsis flu (fluorescent) mutant accumulates a high level of 1O2 and shows drastic changes in nuclear gene expression. Only two plastid proteins, EX1 (executer 1) and EX2 (executer 2), have been identified in the singlet oxygen signaling. Here, we found that the transcription factor abscisic acid insensitive 4 (ABI4) binds the promoters of genes responsive to 1O2-signals. Inactivation of the ABI4 protein in the flu/abi4 double mutant was sufficient to compromise the changes of almost all 1O2-responsive-genes and rescued the lethal phenotype of flu grown under light/dark cycles, similar to the flu/ex1/ex2 triple mutant. In addition to cell death, we reported for the first time that 1O2 also induces cell wall thickening and stomatal development defect. Contrastingly, no apparent growth arrest was observed for the flu mutant under normal light/dim light cycles, but the cell wall thickening (doubled) and stomatal density reduction (by two-thirds) still occurred. These results offer a new idea for breeding stress tolerant plants.

C-C chemokine receptor 5 is essential for conventional NK cell trafficking and liver injury in a murine hepatitis virus-induced fulminant hepatic failure model.

BACKGROUND: Previous studies have demonstrated that natural killer (NK) cells migrated into the liver from peripheral organs and exerted cytotoxic effects on hepatocytes in virus-induced liver failure. AIM: This study aimed to investigate the potential therapeutic role of chemokine receptors in the migration of NK cells in a murine hepatitis  virus strain 3 (MHV-3)-induced fulminant hepatic failure (MHV-3-FHF) model and its mechanism. RESULTS: By gene array analysis, chemokine (C-C motif) receptor 5 (CCR5) was found to have remarkably elevated expression levels in hepatic NK cells after MHV-3 infection. The number of hepatic CCR5+ conventional NK (cNK) cells increased and peaked at 48 h after MHV-3 infection, while the number of hepatic resident NK (rNK) cells steadily declined. Moreover, the expression of CCR5-related chemokines, including macrophage inflammatory protein (MIP)-1α, MIP-1ß and regulated on activation, normal T-cell expressed and secreted (RANTES) was significantly upregulated in MHV-3-infected hepatocytes. In an in vitro Transwell migration assay, CCR5-blocked splenic cNK cells showed decreased migration towards MHV-3-infected hepatocytes, and inhibition of MIP-1ß or RANTES but not MIP-1α decreased cNK cell migration. Moreover, CCR5 knockout (KO) mice displayed reduced infiltration of hepatic cNK cells after MHV-3 infection, accompanied by attenuated liver injury and improved mouse survival time. Adoptive transfer of cNK cells from wild-type mice into CCR5 KO mice resulted in the abundant accumulation of hepatic cNK cells and aggravated liver injury. Moreover, pharmacological inhibition of CCR5 by maraviroc reduced cNK cell infiltration in the liver and liver injury in the MHV-3-FHF model. CONCLUSION: The CCR5-MIP-1ß/RANTES axis played a critical role in the recruitment of cNK cells to the liver during MHV-3-induced liver injury. Targeted inhibition of CCR5 provides a therapeutic approach to ameliorate liver damage during virus-induced acute liver injury.

Characterization of immunomodulatory factors and cells in bronchoalveolar lavage fluid for immune checkpoint inhibitor-related pneumonitis.

As immune checkpoint inhibitors (ICIs) are widely used, a series of immune-related adverse events (irAEs) have been reported, including immune checkpoint inhibitor-related pneumonitis (ICI-pneumonitis). The incidence of ICI-pneumonitis is higher in reality than in clinical trials. The diagnosis is challenging, mainly based on clinical and imaging features, and requires the exclusion of other causes. The data on the biological mechanisms of ICI-pneumonitis are scarce, resulting in little knowledge of the best treatment for ICI-pneumonitis. Bronchoalveolar lavage (BAL) may be helpful to identify the biological differences or find predictive biomarkers, and may in turn help to develop phenotype-specific targeted drugs to treat ICI-pneumonitis. Herein, we outline the characterization of immunomodulatory factors and cells in bronchoalveolar lavage fluid for ICI-pneumonitis. Through careful sorting and literature review, we find crosstalk between pathogenic Th17/Th1 cells (i.e., Th17.1) and pro-inflammatory monocytes, and activation of Th17(/Th1)/IL-17A (/IFN-γ) pathways may play a key role in the pathogenesis of ICI-pneumonitis. Disruption of the interaction between pathogenic Th17/Th1 cells and pro-inflammatory monocytes (such as, anti-IL-23) may be a potential treatment for ICI-pneumonitis. We first describe the possible pathophysiological mechanisms of ICI-pneumonitis, hoping to contribute to the optimization of diagnosis and treatment, as well as provide readers with research inspiration.

Lactose and Galactose Promote the Crystallization of Human Galectin-10.

Galectin-10 (Gal-10) forms Charcot-Leyden crystals (CLCs), which play a key role in the symptoms of asthma and allergies and some other diseases. Gal-10 has a carbohydrate-binding site; however, neither the Gal-10 dimer nor the CLCs can bind sugars. To investigate the monomer-dimer equilibrium of Gal-10, high-performance size-exclusion chromatography (SEC) was employed to separate serial dilutions of Gal-10 with and without carbohydrates. We found that both the dimerization and crystallization of Gal-10 were promoted by lactose/galactose binding. A peak position shift for the monomer was observed after treatment with either lactose or galactose, implying that the polarity of the monomer was reduced by lactose/galactose binding. Further experiments indicated that alkaline conditions of pH 8.8 mimicked the lactose/galactose-binding environment, and the time interval between monomers and dimers in the chromatogram decreased from 0.8 min to 0.4 min. Subsequently, the electrostatic potential of the Gal-10 monomers was computed. After lactose/galactose binding, the top side of the monomer shifted from negatively charged to electrically neutral, allowing it to interact with the carbohydrate-binding site of the opposing subunit during dimerization. Since lactose/galactose promotes the crystallization of Gal-10, our findings implied that dairy-free diets (free of lactose/galactose) might be beneficial to patients with CLC-related diseases.

Different Photosynthetic Response to High Light in Four Triticeae Crops.

Photosynthetic capacity is usually affected by light intensity in the field. In this study, photosynthetic characteristics of four different Triticeae crops (wheat, triticale, barley, and highland barley) were investigated based on chlorophyll fluorescence and the level of photosynthetic proteins under high light. Compared with wheat, three cereals (triticale, barley, and highland barley) presented higher photochemical efficiency and heat dissipation under normal light and high light for 3 h, especially highland barley. In contrast, lower photoinhibition was observed in barley and highland barley relative to wheat and triticale. In addition, barley and highland barley showed a lower decline in D1 and higher increase in Lhcb6 than wheat and triticale under high light. Furthermore, compared with the control, the results obtained from PSII protein phosphorylation showed that the phosphorylation level of PSII reaction center proteins (D1 and D2) was higher in barley and highland barley than that of wheat and triticale. Therefore, we speculated that highland barley can effectively alleviate photodamages to photosynthetic apparatus by high photoprotective dissipation, strong phosphorylation of PSII reaction center proteins, and rapid PSII repair cycle under high light.

Melatonin improves the removal and the reduction of Cr(VI) and alleviates the chromium toxicity by antioxidative machinery in Rhodobacter sphaeroides.

Bioremediation with photosynthetic bacteria (PSB) is thought to be a promising removal method for hexavalent chromium [Cr(VI)]-containing wastewater. In the present study, Rhodobacter sphaeroides (R. sphaeroides) SC01 was used for the investigation of Cr(VI) removal in Cr(VI)-contaminated solution in the presence of melatonin. It was found that exogenous melatonin alleviated oxidative damage to R. sphaeroides SC01, increased Cr (VI) absorption capacity of cell membrane, and improved the reduction efficiency of Cr(VI) via the activation of chromate reductants. The results showed that melatonin could further promote the increase in Cr(VI) removal efficiency, reaching up to 97.8%. Furthermore, melatonin application resulted in 296.9%, 44.4%, and 69.7% upregulation of ascorbic acid (AsA), glutathione (GSH), and cysteine (Cys) relative to non-melatioin treated R. sphaeroides SC01 at 48 h. In addition, the resting cells, cell-free supernatants (CFS), and cell-free extracts (CFE) with melatonin had a higher Cr(VI) removal rate of 18.6%, 82.0%, and 15.2% compared with non-melatonin treated R. sphaeroides SC01. Fourier transform infrared spectroscopy (FTIR) revealed that melatonin increased the binding of Cr(III) with PO43- and CO groups on cell membrane of R. sphaeroides SC01. X-ray diffractometer (XRD) analysis demonstrated that melatonin remarkably bioprecipitated the production of CrPO4·6H2O in R. sphaeroides SC01. Hence, these results indicated that melatonin plays the important role in the reduction and uptake of Cr(VI), demonstrating it is a great promising strategy for the management of Cr(VI) contaminated wastewater in photosynthetic bacteria.

Photosystems and antioxidative system of rye, wheat and triticale under Pb stress.

Lead (Pb2+) pollution in the soil sub-ecosystem has been a continuously growing problem due to economic development and ever-increasing anthropogenic activities across the world. In this study, the photosynthetic performance and antioxidant capacity of Triticeae cereals (rye, wheat and triticale) were compared to assess the activities of antioxidants, the degree of oxidative damage, photochemical efficiency and the levels of photosynthetic proteins under Pb stress (0.5 mM, 1 mM and 2 mM Pb (NO3)2). Compared with triticale, Pb treatments imposed severe oxidative damage in rye and wheat. In addition, the highest activity of major antioxidant enzymes (SOD, POD, CAT, and GPX) was also found to be elevated. Triticale accumulated the highest Pb contents in roots. The concentration of mineral ions (Mg, Ca, and K) was also high in its leaves, compared with rye and wheat. Consistently, triticale showed higher photosynthetic activity under Pb stress. Immunoblotting of proteins revealed that rye and wheat have significantly lower levels of D1 (photosystem II subunit A, PsbA) and D2 (photosystem II subunit D, PsbD) proteins, while no obvious decrease was noticed in triticale. The amount of light-harvesting complex II b6 (Lhcb6; CP24) and light-harvesting complex II b5 (Lhcb5; CP26) was significantly increased in rye and wheat. However, the increase in PsbS (photosystem II subunit S) protein only occurred in wheat and triticale exposed to Pb treatment. Taken together, these findings demonstrate that triticale shows higher antioxidant capacity and photosynthetic efficiency than wheat and rye under Pb stress, suggesting that triticale has high tolerance to Pb and could be used as a heavy metal-tolerant plant.

Chloroplastic photoprotective strategies differ between bundle sheath and mesophyll cells in maize (Zea mays L.) Under drought.

Bundle sheath cells play a crucial role in photosynthesis in C4 plants, but the structure and function of photosystem II (PSII) in these cells is still controversial. Photoprotective roles of bundle sheath chloroplasts at the occurrence of environmental stresses have not been investigated so far. Non-photochemical quenching (NPQ) of chlorophyll a fluorescence is the photoprotective mechanism that responds to a changing energy balance in chloroplasts. In the present study, we found a much higher NPQ in bundle sheath chloroplasts than in mesophyll chloroplasts under a drought stress. This change was accompanied by a more rapid dephosphorylation of light-harvesting complex II (LHCII) subunits and a greater increase in PSII subunit S (PsbS) protein abundance than in mesophyll cell chloroplasts. Histochemical staining of reactive oxygen species (ROS) suggested that the high NPQ may be one of the main reasons for the lower accumulation of ROS in bundle sheath chloroplasts. This may maintain the stable functioning of bundle sheath cells under drought condition. These results indicate that the superior capacity for dissipation of excitation energy in bundle sheath chloroplasts may be an environmental adaptation unique to C4 plants.

Exogenous melatonin improved photosynthetic efficiency of photosystem II by reversible phosphorylation of thylakoid proteins in wheat under osmotic stress.

It has been well demonstrated that melatonin plays an important protective role in photosynthesis of plants under various environmental stresses, while the detailed mechanisms by which melatonin protects photosystem II (PSII) under environmental stress are still unclear. In the study, the effects of melatonin on photosynthetic efficiency, energy dissipation, PSII protein composition, and reversible phosphorylation of thylakoid proteins were investigated in wheat plants under osmotic stress. The results showed that osmotic stress significantly reduced pigment content, photochemical efficiency of PSII, oxygen-evolving activity, and dissipation of excess excitation energy, while 25 µM melatonin applications greatly alleviated their decline under osmotic stress. Western blot data of PSII proteins revealed that melatonin upregulated the levels of D1, Lhcb5, Lhcb6, PsbQ, and PsbS proteins in wheat exposed to osmotic stress. In addition, thylakoid membrane proteins were strongly phosphorylated in wheat under osmotic stress with or without melatonin. Furthermore, the results from PSII protein dephosphorylation showed that exogenous melatonin promoted the dephosphorylation of LCHII, CP43, and D1 under osmotic stress. Therefore, our findings suggest that melatonin can provide an effective protection for the photosynthetic apparatus by the regulation of PSII proteins and the reversible phosphorylation of thylakoid proteins under drought stress.

Beneficial Effects of Sodium Nitroprusside on the Aroma, Flavors, and Anthocyanin Accumulation in Blood Orange Fruits.

The quality of Tarocco blood orange (Citrus sinensis (L.) Osbeck), which has been cultivated for many years, has degraded substantially. Decreased sugar content, decreased blood color, and increased sour flavor have developed as a result. To improve fruit quality, we studied the effects of bagging and sodium nitroprusside, as a nitric oxide (NO) donor, on the fruit quality of Tarocco blood orange two months before picking. The results showed that NO treatment effectively improved the content of total soluble solids and limonene in the fruit, as well as the color and hardness of the fruit, but reduced the tannin content. It also increased the contents of soluble sugar, fructose, sucrose, vitamin C, amino acids, and mineral elements. NO treatment inhibited the activities of polygalacturonase and pectin esterase, delayed the degradation of protopectin, and promoted the accumulation of anthocyanins, total flavonoids, and flavonoids synthesis. Thus, NO treatment improved the aroma, flavors, and physical properties of blood orange fruit.

Abuse of amantadine in poultry may be associated with higher fatality rate of H5N1 infections in humans.

Amantadine, an antiviral drug, has been widely used in human anti-influenza treatments. However, several highly pathogenic avian influenza viruses show amantadine-resistance mutations in the viral matrix 2 (M2) protein. Here we analyzed global H5N1 sequencing data and calculate possible correlations between frequencies of key mutations in M2 and the mortality rates. We found that the frequency of L26I/V27A mutation in M2 (isolated from both human and avian hosts) is linearly correlated with the mortality rates of human H5N1 infections. The significant correlation between M2 mutations in avians and the mortality rates in humans suggests that the pre-existence of L26I/V27A in birds may determine patient fatalities after transinfections from avian to human hosts. 100% prevalence of L26I/V27A mutation increased the mortality rates from 51% (95% confidence interval [CI] 37%-65%) to 89% (95% CI 88%-90%). Mutations involving Leu26 or Val27 were identified to be the major mutations emerging from drug selection pressure. Thus the emergence of the super H5N1 virus with a fatality of over 90% may be attributed to the abuse of amantadine in poultry, especially in some southeast Asian countries. A more stringent control to antiviral veterinary drugs is imperative.