A reporter for noninvasively monitoring gene expression and plant transformation

Reporters have been widely used to visualize gene expression,protein localization,and other cellular activities,but the commonly used reporters require special equipment,expensive chemicals,or invasive treatments.Here,we construct a new reporter RUBY that converts tyrosine to vividly red betalain,which is clearly visible to naked eyes without the need of using special equipment or chemical treatments.We show that RUBY can be used to noninvasively monitor gene expression in plants.Furthermore,we show that RUBY is an effective selection marker for transformation events in both rice and Arabidopsis.The new reporter will be especially useful for monitoring cellular activities in large crop plants such as a fruit tree under field conditions and for observing transformation and gene expression in tissue culture under sterile conditions.

Efficacy of SpyGlass^(TM)-directed biopsy compared to brush cytology in obtaining adequate tissue for diagnosis in patients with biliary strictures

AIM: To evaluate the diagnostic yield(inflammatory activity) and efficiency(size of the biopsy specimen) of SpyGlassTM-guided biopsy vs standard brush cytology in patients with and without primary sclerosing cholangitis(PSC).METHODS: At the University Medical Center Mainz, Germany, 35 consecutive patients with unclear biliarylesions(16 patients) or long-standing PSC(19 patients) were screened for the study. All patients underwent a physical examination, lab analyses, and abdominal ultrasound. Thirty-one patients with non-PSC strictures or with PSC were scheduled to undergo endoscopic retrograde cholangiography(ERC) and subsequent per-oral cholangioscopy(POC). Standard ERC was initially performed, and any lesions or strictures were localized. POC was performed later during the same session. The Boston Scientific SpyGlass SystemTM(Natick, MA, United States) was used for choledochoscopy. The biliary tree was visualized, and suspected lesions or strictures were biopsied, followed by brush cytology of the same area. The study endpoints(for both techniques) were the degree of inflammation, tissue specimen size, and the patient populations(PSC vs non-PSC). Inflammatory changes were divided into three categories: none, low activity, and high activity. The specimen quantity was rated as low, moderate, or sufficient.RESULTS: SpyGlassTM imaging and brush cytology with material retrieval were performed in 29 of 31(93.5%) patients(23 of the 29 patients were male). The median patient age was 45 years(min, 20 years; max, 76 years). Nineteen patients had known PSC, and 10 showed non-PSC strictures. No procedure-related complications were encountered. However, for both methods, tissues could only be retrieved from 29 pa-tients. In cases of inflammation of the biliary tract, the diagnostic yield of the SpyGlassTM-directed biopsies was greater than that using brush cytology. More tissue material was obtained for the biopsy method than for the brush cytology method(P = 0.021). The biopsies showed significantly more inflammatory characteristics and greater inflammatory activity compared to the cy-tological investigation(P = 0.014). The greater quantity of tissue samples proved useful for both PSC and non-PSC patients.CONCLUSION: SpyGlassTM imaging can be recom-mended for proper inflammatory diagnosis in PSC pa-tients. However, its value in diagnosing dysplasia wasnot addressed in this study and requires further investi-gation.

Induced pluripotent stem cells from Huntington's disease patients:a promising approach to define and correct disease-related alterations

Adult somatic cells such as skin or blood cells from either health donors or patients can be reprogrammed into induced pluripotent stem cells(iPSCs).Given their unlimited self-renewal and differentiation capacities,iPSCs are an invaluable resource to generate terminally differentiated cells.Thus,iPSCs can facilitate the study of human diseases and drug screening,holding great promise for regenerative medicine.Another significant advantage of iPSC disease-modeling is that normal and mutant proteins are expressed at endogenous levels.In addition,subtle phenotypes and the effects of genetic background variations can be assessed by comparison between iPSC lines obtained from different patients and healthy donors as well as isogenic lines,in which disease-related mutations are corrected.

COVID-19's impacts on dengue transmission:Focus on neighbourhood surveillance of Aedes mosquitoes

The ongoing COVID-19 pandemic has impacted the entire globe on all fronts,and vector-borne diseases are not an exception.There are certain similarities between dengue and COVID-19 since both diseases are positive-sense single-stranded RNA virus and have animal origin linkages.Interestingly,both the diseases present over 80%asymptomatic cases.Dengue is the most prevalent and fast-emerging viral infection worldwide.The dengue virus(DENV)has four serotypes,namely DENV-1,DENV-2,DENV-3 and DENV-4,and it is possible that the same person can be infected four times before full immunity is established[1];whereas,COVID-19 is an air-borne respiratory disease caused by the severe acute respiratory syndrome coronavirus 2,and several variants have emerged over time.In late 2020,the variants posed an increased risk to global public health emergency,which prompted the characterisation of specific Variants of Interest and Variants of Concern to mitigate the COVID-19 pandemic[2].DENV is transmitted by several species of day-biting Aedes mosquitoes.They are highly adaptive and invasive species and are predominantly found in the tropical and subtropical regions.In recent decades,these mosquitoes have been discovered in all continents except Antarctica.Aedes aegypti and Aedes albopictus are two major vectors of dengue.This means wherever these mosquitoes make their footprints,dengue creeps in.

Somatostatin-Positive Neurons in the Rostral Zona Incerta Modulate Innate Fear-Induced Defensive Response in Mice

Defensive behaviors induced by innate fear or Pavlovian fear conditioning are crucial for animals to avoid threats and ensure survival.The zona incerta(ZI)has been demonstrated to play important roles in fear learning and fear memory,as well as modulating auditory-induced innate defensive behavior.However,whether the neuronal subtypes in the ZI and specific circuits can mediate the innate fear response is largely unknown.Here,we found that somatostatin(SST)-positive neurons in the rostral ZI of mice were activated by a visual innate fear stimulus.Optogenetic inhibition of SST-positive neurons in the rostral ZI resulted in reduced flight responses to an overhead looming stimulus.Optogenetic activation of SST-positive neurons in the rostral ZI induced fear-like defensive behavior including increased immobility and bradycardia.In addition,we demonstrated that manipulation of the GABAergic projections from SST-positive neurons in the rostral ZI to the downstream nucleus reuniens(Re)mediated fear-like defensive behavior.Retrograde trans-synaptic tracing also revealed looming stimulus-activated neurons in the superior colliculus(SC)that projected to the Re-projecting SST-positive neurons in the rostral ZI(SC-ZIrSST-Re pathway).Together,our study elucidates the function of SST-positive neurons in the rostral ZI and the SC-ZIrSST-Re tri-synaptic circuit in mediating the innate fear response.

Induction of Anxiety-Like Phenotypes by Knockdown of Cannabinoid Type-1 Receptors in the Amygdala of Marmosets

The amygdala is an important hub for regulating emotions and is involved in the pathophysiology of many mental diseases,such as depression and anxiety.Meanwhile,the endocannabinoid system plays a crucial role in regulating emotions and mainly functions through the cannabinoid type-1 receptor(CB1R),which is strongly expressed in the amygdala of non-human primates(NHPs).However,it remains largely unknown how the CB1Rs in the amygdala of NHPs regulate mental diseases.Here,we investigated the role of CB1R by knocking down the cannabinoid receptor 1(CNR1)gene encoding CB1R in the amygdala of adult marmosets through regional delivery of AAV-SaCas9-gRNA.We found that CB1R knockdown in the amygdala induced anxiety-like behaviors,including disrupted night sleep,agitated psychomotor activity in new environments,and reduced social desire.Moreover,marmosets with CB1R-knockdown had up-regulated plasma cortisol levels.These results indicate that the knockdown of CB1Rs in the amygdala induces anxiety-like behaviors in marmosets,and this may be the mechanism underlying the regulation of anxiety by CB1Rs in the amygdala of NHPs.

Whole-Brain Map of Long-Range Monosynaptic Inputs to Different Cell Types in the Amygdala of the Mouse

The amygdala,which is involved in various behaviors and emotions,is reported to connect with the whole brain.However,the long-range inputs of distinct cell types have not yet been defined.Here,we used a retrograde trans-synaptic rabies virus to generate a whole-brain map of inputs to the main cell types in the mouse amygdala.We identified 37 individual regions that projected to neurons expressing vesicular glutamate transporter 2,78 regions to parvalbumin-expressing neurons,104 regions to neurons expressing protein kinase C-δ,and 89 regions to somatostatin-expressing neurons.The amygdala received massive projections from the isocortex and striatum.Several nuclei,such as the caudate-putamen and the CA1 field of the hippocampus,exhibited input preferences to different cell types in the amygdala.Notably,we identified several novel input areas,including the substantia innominata and zona incerta.These findings provide anatomical evidence to help understand the precise connections and diverse functions of the amygdala.

Effect of Apolipoprotein E Genotypes on Huntington’s Disease Phenotypes in a Han Chinese Population

Huntington's disease(HD)is an autosomal dominant degenerative disease that mainly encompasses movement,cognition,and behavioral symptoms.The apolipoprotein E(APOE)gene is thought to be associated with many neurodegenerative diseases.Here,we enrolled a cohort of 223 unrelated Han Chinese patients with HD and1241 unrelated healthy controls in Southeastern China and analyzed the correlation between APOE genotypes and HD phenotypes.The results showed that the frequency of the E4 allele(7.1%)in HD patients was statistically less than that in controls(12.0%)(P =0.004).In addition,we divided patients into motor-onset and non-motor-onset groups,and analyzed the relationship with APOE genotypes.The results,however,were negative.Furthermore,the age at onset(AAO),defined as the age at the onset of motor symptoms,was compared in each APOE genotype subgroup and multivariate regression analysis was used to exclude the interference of CAG repeat length on AAO,but no association was found between APOE genotypes and AAO.Finally,we analyzed adult-onset HD to exclude the interference caused by juvenile HD(n = 13),and the results were negative.Therefore,our study suggests that APOE may not be a genetic modifier for HD,especially for adult-onset HD among Chinese of Han ethnicity.To the best of our knowledge,this is the first study of the correlation between APOE genotypes and HD phenotypes in a Han Chinese population.

Correlation Between CCG Polymorphisms and CAG Repeats During Germline Transmission in Chinese Patients with Huntington’s Disease

Dear Editor,Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease characterized by movement disorder,progressive dementia,and psychiatric and behavioral changes.It is caused by unstable expanded CAG trinucleotide repeats in exon 1 of the huntingtin (HTT)gene,located on chromosome 4p16.3 [1].

Pathological Networks Involving Dysmorphic Neurons in Type ⅡFocal Cortical Dysplasia

Focal cortical dysplasia(FCD)is one of the most common causes of drug-resistant epilepsy.Dysmorphic neurons are the major histopathological feature of typeⅡFCD,but their role in seizure genesis in FCD is unclear.Here we performed whole-cell patch-clamp recording and morphological reconstruction of cortical principal neurons in postsurgical brain tissue from drug-resistant epilepsy patients.Quantitative analyses revealed distinct morphological and electrophysiological characteristics of the upper layer dysmorphic neurons in typeⅡFCD,including an enlarged soma,aberrant dendritic arbors,increased current injection for rheobase action potential firing,and reduced action potential firing frequency.Intriguingly,the upper layer dysmorphic neurons received decreased glutamatergic and increased GABAergic synaptic inputs that were coupled with upregulation of the Na^(+)-K^(+)-Cl^(−)cotransporter.In addition,we found a depolarizing shift of the GABA reversal potential in the CamKⅡ-cre::PTENflox/flox mouse model of drug-resistant epilepsy,suggesting that enhanced GABAergic inputs might depolarize dysmorphic neurons.Thus,imbalance of synaptic excitation and inhibition of dysmorphic neurons may contribute to seizure genesis in typeⅡFCD.

iGMDR:Integrated Pharmacogenetic Resource Guide to Cancer Therapy and Research

Current pharmacogenetic studies have obtained many genetic models that can predict the therapeutic efficacy of anticancer drugs.Although some of these models are of crucial importance and have been used in clinical practice,these very valuable models have not been well adopted into cancer research to promote the development of cancer therapies due to the lack of integration and standards for the existing data of the pharmacogenetic studies.For this purpose,we built a resource investigating genetic model of drug response(iGMDR),which integrates the models from in vitro and in vivo pharmacogenetic studies with different omics data from a variety of technical systems.In this study,we introduced a standardized process for all integrations,and described how users can utilize these models to gain insights into cancer.iGMDR is freely accessible at https://igmdr.modellab.cn.

Clinical features and genetic spectrum in Chinese patients with recessive hereditary spastic paraplegia

Background:Although many causative genes of hereditary spastic paraplegia(HSP)have been uncovered in recent years,there are still approximately 50% of HSP patients without genetically diagnosis,especially in autosomal recessive(AR)HSP patients.Rare studies have been performed to determine the genetic spectrum and clinical profiles of recessive HSP patients in the Chinese population.Methods:In this study,we investigated 24 Chinese index AR/sporadic patients by targeted next-generation sequencing(NGS),Sanger sequencing and multiplex ligation-dependent probe amplification(MLPA).Further functional studies were performed to identify pathogenicity of those uncertain significance variants.Results:We identified 11 mutations in HSP related genes including 7 novel mutations,including two(p.V1979_L1980delinsX,p.F2343 fs)in SPG11,two(p.T55 M,p.S308 T)in AP5Z1,one(p.S242N)in ALDH18A1,one(p.D597fs)in GBA2,and one(p.Q486X)in ATP13A2 in 8 index patients and their family members.Mutations in ALDH18A1,AP5Z1,CAPN1 and ATP13A2 genes were firstly reported in the Chinese population.Furthermore,the clinical phenotypes of the patients carrying mutations were described in detail.The mutation(p.S242 N)in ALDH18A1 decreased enzyme activity of P5CS and mutations(p.T55 M,p.S308 T)in AP5Z1 induced lysosomal dysfunction.Conclusion:Our results expanded the genetic spectrum and clinical profiles of AR-HSP patients and further demonstrated the efficiency and reliability of targeted NGS diagnosing suspected HSP patients.

Cytokine Profiling in Influenza A Virus and Staphylococcal(Co-)Infections

Influenza A virus and Staphylococcus aureus are common causative agents of pneumonia.Co-infections with these two pathogens frequently occur and are characterized,among others,by higher morbidity and mortality due to hyper-inflammation of the lungs.Here,we aimed to profile systemic and local cytokine composition at early acute stages of pneumonia in amurinemodel.Allmice recovered from single influenza A virus and/or staphylococcal infections.In contrast,co-infections led to a severe clinical outcome.While distinct cytokine patterns were detected in lungs of single-pathogen-infected animals,co-infections combined both virus-and bacteria-driven responses.However,analyses of infected human primarymonocytic cells as well as bronchial epithelial cells did not reflectmurine profiles.Based on infectious dose,mainly bacteria-driven responses were noted.The impact of single cells to cytokine composition of the lungs and translation of murine studies to humans remains uncertain and warrants further studies.

A Deep Mesencephalic Nucleus Circuit Regulates Licking Behavior

Licking behavior is important for water intake.The deep mesencephalic nucleus(DpMe)has been implicated in instinctive behaviors.However,whether the DpMe is involved in licking behavior and the precise neural circuit behind this behavior remains unknown.Here,we found that the activity of the DpMe decreased during water intake.Inhibition of vesicular glutamate transporter 2-positive(VGLUT2+)neurons in the DpMe resulted in increased water intake.Somatostatin-expressing(SST+),but not protein kinase C-expressing(PKC-8+),GABAergic neurons in the central amygdala(CeA)preferentially innervated DpMe VGLUT2+neurons.The SST+neurons in the CeA projecting to the DpMe were activated at the onset of licking behavior.Activation of these CeA SST+GABAergic neurons,but not PKC-8+GABAergic neurons,projecting to the DpMe was sufficient to induce licking behavior and promote water intake.These findings redefine the roles of the DpMe and reveal a novel CeAssT_DpMevcLUT?cireuit that regulaes icking behavior and promotes water intake.