Investigating the expression profiles of cysteine string proteins(CSPs)in cochlear tissue

Objective:This study aims to explore the expression patterns of cysteine string protein alpha(CSPα)and cysteine string protein beta(CSPβ)in the mammalian inner ear,with an emphasis on their temporal dynamics during the developmental stages of C57BL/6 mice.Methods:We utilized immunofluorescence staining to assess the localization and distribution of CSPαand CSPβwithin the inner ears of C57BL/6 mice and miniature pigs.Additionally,this method facilitated the investigation of their temporal expression profiles.Results:In adult C57BL/6 mice and miniature pigs,CSPαand CSPβwere identified in the cytoplasm of inner hair cells and spiral ganglion cells,yet were absent in outer hair cells.Both proteins were found to colocalize with Ctbp2 on the basal side of the cytoplasm in inner hair cells’basilar membrane.Expression of CSPαwas observed at the nerve fiber termini at the basilar membrane’s base of inner and outer hair cells 10 days postnatally in C57BL/6 mice.Notably,expression of both CSPαand CSPβin the cytoplasm of inner hair cells emerged on the 12th day post-birth,aligning with the timeline for registering cochlear potentials.The expression levels of both proteins increased with age,but were consistently absent in outer hair cells.Contrastingly,expression of CSPαand CSPβwas present in the cytoplasm of inner hair cells in miniature pigs as early as one day post-birth,yet remained absent in the three rows of outer hair cells.Conclusion:CSPαand CSPβexhibit predominant and specific expression in inner hair cells and spiral ganglion cells.A unique expression pattern was observed for CSPα,which was also present at the nerve fiber endings of both inner and outer hair cells.The developmental expression trajectory of CSPαand CSPβin mouse inner hair cells is characterized by an initial absence,followed by a gradual increase.Moreover,the timing of expression onset between mice and miniature pigs indicates distinct temporal dynamics,suggesting a potential role in auditory development.

Discovery of novel phosphodiesterase-1 inhibitors for curing vascular dementia:Suppression of neuroinflammation by blocking NF-κB transcription regulation and activating cAMP/CREB axis

Vascular dementia(VaD)is the second commonest type of dementia which lacks of efficient treatments currently.Neuroinflammation as a prominent pathological feature of VaD,is highly involved in the development of VaD.In order to verify the therapeutic potential of PDE1 inhibitors against VaD,the anti-neuroinflammation,memory and cognitive improvement were evaluated in vitro and in vivo by a potent and selective PDE1 inhibitor 4a.Also,the mechanism of 4a in ameliorating neuroinflammation and VaD was systematically explored.Furthermore,to optimize the drug-like properties of 4a,especially for metabolic stability,15 derivatives were designed and synthesized.As a result,candidate 5f,with a potent IC50 value of 4.5 nmol/L against PDE1C,high selectivity over PDEs,and remarkable metabolic stability,efficiently ameliorated neuron degeneration,cognition and memory impairment in VaD mice model by suppressing NF-κB transcription regulation and activating cAMP/CREB axis.These results further identified PDE1 inhibition could serve as a new therapeutic strategy for treatment of VaD.

In vitro and in vivo efficacy of Molnupiravir against Zika virus infections

Dear Editor,Zika virus(ZIKV)is a mosquito-borne,positive-stranded RNA virus first identified in 1947 in monkeys and later identified in humans in 1952(Faye et al.,2014).It is among the“TORCH”group of microorganisms and causes outbreaks in several countries and regions since 2007(Voordouw et al.,2019).

Antiviral activity of vitamin D derivatives against severe fever with thrombocytopenia syndrome virus in vitro and in vivo

Severe fever with thrombocytopenia syndrome virus(SFTSV)is a tick-borne virus that causes the severe fever thrombocytopenia syndrome,which manifests as fever and haemorrhage,accompanied by severe neurological complications.To date,no specific antiviral drugs have been approved for this indication.Herein,we investigated whether vitamin D derivatives inhibit SFTSV both in vitro and in vivo.An in vitro study demonstrated that vitamin D derivatives significantly suppressed viral RNA replication,plaque formation,and protein expression in a dosedependent manner.Subsequently,in vivo studies revealed that doxercalciferol and alfacalcidol were associated with increased survival and reduced viral RNA load in the blood.Time-of-addition assay suggested that vitamin D derivatives primarily acted during the post-entry phase of SFTSV infection.However,cytopathic effect protective activity was not observed in RIG-I immunodeficient cell line Huh7.5,and the administration of vitamin D derivatives did not improve the survival rates or reduce the blood viral loads in adult A129 mice.Further transcriptome exploration into the antiviral mechanism revealed that alfacalcidol stimulates host innate immunity to exert antiviral effects.To expand the application of vitamin D derivatives,in vitro and in vivo drug combination assays were performed,which highlighted the synergistic effects of vitamin D derivatives and T-705 on SFTSV.The combination of alfacalcidol and T-705 significantly enhanced the therapeutic effects in mice.This study highlights the potential of vitamin D derivatives against SFTSV and suggests that they may have synergistic effects with other compounds used in the treatment of SFTSV infection.

Discovery of highly potent phosphodiesterase-1 inhibitors by a combined-structure free energy perturbation approach

Accurate receptor/ligand binding free energy calculations can greatly accelerate drug discov-ery by identifying highly potent ligands.By simulating the change from one compound structure to another,the relative binding free energy(RBFE)change can be calculated based on the theoretically rigorous free energy perturbation(FEP)method.However,existing FEP-RBFE approaches may face convergence challenges due to difficulties in simulating non-physical intermediate states,which can lead to increased computational costs to obtain the converged results.To fundamentally overcome these issues and accelerate drug discovery,a new combined-structure RBFE(CS-FEP)calculation strategy was proposed,which solved the existing issues by constructing a new alchemical pathway,smoothed the alchemical transformation,increased the phase-space overlap between adjacent states,and thus signif-icantly increased the convergence and accelerated the relative binding free energy calculations.This method was extensively tested in a practical drug discovery effort by targeting phosphodiesterase-1(PDE1).Starting from a PDE1 inhibitor(compound 9,IC_(50)=16.8 mmol/L),the CS-FEP guided hit-to-lead optimizations resulted in a promising lead(11b and its mesylate salt formulation 11b-Mesylate,IC_(50)=7.0 nmol/L),with w2400-fold improved inhibitory activity.Further experimental studies re-vealed that the lead showed reasonable metabolic stability and significant anti-fibrotic effects in vivo.