Effects of P301L-TAU on post-translational modifications of microtubules in human iPSC-derived cortical neurons and TAU transgenic mice

TAU is a microtubule-associated protein that promotes microtubule assembly and stability in the axon.TAU is missorted and aggregated in an array of diseases known as tauopathies.Microtubules are essential for neuronal function and regulated via a complex set of post-translational modifications,changes of which affect microtubule stability and dynamics,microtubule interaction with other proteins and cellular structures,and mediate recruitment of microtubule-severing enzymes.As impairment of microtubule dynamics causes neuronal dysfunction,we hypothesize cognitive impairment in human disease to be impacted by impairment of microtubule dynamics.We therefore aimed to study the effects of a disease-causing mutation of TAU(P301L)on the levels and localization of microtubule post-translational modifications indicative of microtubule stability and dynamics,to assess whether P301L-TAU causes stability-changing modifications to microtubules.To investigate TAU localization,phosphorylation,and effects on tubulin post-translational modifications,we expressed wild-type or P301L-TAU in human MAPT-KO induced pluripotent stem cell-derived neurons(i Neurons)and studied TAU in neurons in the hippocampus of mice transgenic for human P301L-TAU(p R5 mice).Human neurons expressing the longest TAU isoform(2N4R)with the P301L mutation showed increased TAU phosphorylation at the AT8,but not the p-Ser-262 epitope,and increased polyglutamylation and acetylation of microtubules compared with endogenous TAU-expressing neurons.P301L-TAU showed pronounced somatodendritic presence,but also successful axonal enrichment and a similar axodendritic distribution comparable to exogenously expressed 2N4R-wildtype-TAU.P301L-TAU-expressing hippocampal neurons in transgenic mice showed prominent missorting and tauopathy-typical AT8-phosphorylation of TAU and increased polyglutamylation,but reduced acetylation,of microtubules compared with non-transgenic littermates.In sum,P301L-TAU results in changes in microtubule PTMs,suggestive of impairment of microtubule stability.This is accompanied by missorting and aggregation of TAU in mice but not in i Neurons.Microtubule PTMs/impairment may be of key importance in tauopathies.

Suppression of mature TAU isoforms prevents Alzheimer's disease-like amyloid-beta oligomer-induced spine loss in rodent neurons

Introduction:TAU isoforms as disease mediators:The microtubule-associated protein TAU is predominantly present in the axons of neurons under physiological conditions.In Alzheimer’s disease(AD)and related tauopathies,TAU also mislocalizes("TAU missorting")to the soma and the dendrites,where it eventually forms aggregates,the so-called neurofibrillary tangles(for review see Zimmer-Bensch and Zempel,2021;Zempel,2023).

Amyloid-β-induced disruption of axon-initial-segment mitochondria localization:consequences for TAU missorting in Alzheimer's disease pathology

TAU is a neuronal microtubule-associated protein preferentially located in axons.In a battery of neurodegenerative diseases termed"tauopathies,"including Alzheimer's disease (AD),TAU is missorted and abnormally phosphorylated,leading to filamentous accumulations of hyperphosphorylated TAU,a pathological hallmark and potential disease driver of AD and related tauopathies (Zempel,2024).

Microtubule affinity regulating kinase(MARK/Par1)isoforms differentially regulate Alzheimer-like TAU missorting and Aβ-mediated synapse pathology

Importance of TAU protein for dementia syndromes:Dementia currently affects about 55 million people worldwide,with Alzheimer's disease(AD)being the most prevalent form.The one crucial pathological hallmark of AD that correlates best with loss of synapses and cognitive decline are the so-called intracellular neurofibrillary tangles composed of mislocalized/missorted and hyperphosphorylated TAU protein(Naseri et al.,2019).Many other neurodegenerative diseases,both genetic and nongenetic,are characterized by neurofibrillary ta ngles or pathological accumulation of the protein TAU and are thus termed"tauopathies".

In situ aneuploidy assessment in human sperm:the use of primed in situ and peptide nucleic acid-fluorescence in situ hybridization techniques

Both the primed in situ(PRINS)and the peptide nucleic acid-fluorescence in situ hybridization(PNA-FISH) techniques constitute alternatives to the conventional(fluorescence in situ hybridization,FISH)procedure for chro- mosomal investigations.The PRINS reaction is based on the use of a DNA polymerase and labeled nucleotide in an in situ primer extension reaction.Peptide nucleic acid probes are synthetic DNA analogs with uncharged polyamide backbones.The two procedures present several advantages(specificity,rapidity and discriminating ability)that make them very attractive for cytogenetic purposes.Their adaptation to human spermatozoa has allowed the development of new and fast procedures for the chromosomal screening of male gametes and has provided efficient complements to FISH for in situ assessment of aneuploidy in male gametes.(Asian J Androl 2006 Jul;8:387-392)

B7 homolog 3 in pancreatic cancer

Despite advances in cancer treatment,pancreatic cancer(PC)remains a disease with high mortality rates and poor survival outcomes.The B7 homolog 3(B7-H3)checkpoint molecule is overexpressed among many malignant tumors,including PC,with low or absent expression in healthy tissues.By modulating various immunological and nonimmunological molecular mechanisms,B7-H3 may influence the progression of PC.However,the impact of B7-H3 on the survival of patients with PC remains a subject of debate.Still,most available scientific data recognize this molecule as a suppressive factor to antitumor immunity in PC.Furthermore,it has been demonstrated that B7-H3 stimulates the migration,invasion,and metastasis of PC cells,and enhances resistance to chemotherapy.In preclinical models of PC,B7-H3-targeting monoclonal antibodies have exerted profound antitumor effects by increasing natural killer cell-mediated antibodydependent cellular cytotoxicity and delivering radioisotopes and cytotoxic drugs to the tumor site.Finally,PC treatment with B7-H3-targeting antibody-drug conjugates and chimeric antigen receptor T cells is being tested in clinical studies.This review provides a comprehensive analysis of all PC-related studies in the context of B7-H3 and points to deficiencies in the current data that should be overcome by future research.

Understanding genetics, sex and signaling: Implications of sex-dependent APOE4-neutrophil-microglia interactions for Alzheimer’s and tauopathies

A recent study published in Nature Medicine uncovers a novel sex-dependent mechanism involving the apolipoprotein E ε4 (APOE4) and in particular neutrophils and their signaling to microglia, which then would mediate cognitive decline in late-onset Alzheimer’s disease (AD).1 We here put the main findings into perspective with current knowledge about APOE4 genotype and neutrophils in the pathogenesis of AD and related tauopathies, and discuss future research directions and therapeutic implications.

Targeting miRNA by CRISPR/Cas in cancer:advantages and challenges

Clustered regulatory interspaced short palindromic repeats(CRISPR)has changed biomedical research and provided entirely new models to analyze every aspect of biomedical sciences during the last decade.In the study of cancer,the CRISPR/CRISPR-associated protein(Cas)system opens new avenues into issues that were once unknown in our knowledge of the non-coding genome,tumor heterogeneity,and precision medicines.CRISPR/Cas-based geneediting technology now allows for the precise and permanent targeting of mutations and provides an opportunity to target small non-coding RNAs such as microRNAs(miRNAs).However,the development of effective and safe cancer gene editing therapy is highly dependent on proper design to be innocuous to normal cells and prevent introducing other abnormalities.This study aims to highlight the cutting-edge approaches in cancer-gene editing therapy based on the CRISPR/Cas technology to target miRNAs in cancer therapy.Furthermore,we highlight the potential challenges in CRISPR/Cas-mediated miRNA gene editing and offer advanced strategies to overcome them.

Serum bile acid profiling reflects enterohepatic detoxification state and intestinal barrier function in inflammatory bowel disease

AIM： To determine free and conjugated serum bile acid （BA） levels in inflammatory bowel disease （IBD） subgroups with defined clinical manifestations. METHODS： Comprehensive serum BA profiling was performed in 358 IBD patients and 310 healthy con- trols by liquid chromatography coupled to electrospray ionization tandem mass spectrometry. RESULTS： Serum levels of hyodeoxycholic acid, the CYP3A4-mediated detoxification product of the second- ary BA lithocholic acid （LCA）, was increased significantly in Crohn＇s disease （CD） and ulcerative colitis （UC）, while most other serum BA species were decreased signifi- cantly. Total BA, total BA conjugate, and total BA glyco- conjugate levels were decreased only in CD, whereas total unconjugated BA levels were decreased only in UC. In UC patients with hepatobiliary manifestations, the conjugated primary BAs glycocholic acid, taurocholic acid, and glycochenodeoxycholic acid were as signifi- cantly increased as the secondary BAs LCA, ursodeoxy- cholic acid, and tauroursodeoxycholic acid compared to UC patients without hepatobiliary manifestations. Finally, we found that in ileocecal resected CD patients, the unconjugated primary BAs, cholic acid and chenode- oxycholic acid, were increased significantly compared to controls and patients without surgical interventions. CONCLUSION： Serum BA profiling in IBD patients that indicates impaired intestinal barrier function and increased detoxification is suitable for advanced diag- nostic characterization and differentiation of IBD sub- groups with defined clinical manifestations.

hsa-mir-183 is frequently methylated and related to poor survival in human hepatocellular carcinoma

To screen clinically relevant microRNAs (miRNAs) silenced by DNA methylation in human hepatocellular carcinoma (HCC).METHODSKnockdown of DNA methyltransferases (DNMTs) using siRNAs and miRNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated miRNA downregulation. Confirmation using individual quantitative real-time PCR (qRT-PCR) assays was then performed followed by DNA methylation quantification at the promoter of the miRNA genes. Quantification of DNA methylation and miRNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables.RESULTSmiRNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of miR-23, miR-25 and miR-183. After qRT-PCR confirmation and CpG island methylation quantification of these miRNAs in cell lines, further analysis in primary HCC specimens showed that hsa-miR-183 is hypermethylated in 30% of HCC (n = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, DNMT knockdown and 5-aza-2’-deoxycytidine treatment reduced methylation and stimulated expression of miR-183. In HCC patients, hypermethylation at hsa-miR-183 promoter significantly correlates with poor survival (log-rank test P = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at hsa-miR-183 is specific for the malignant transformation of hepatocytes.CONCLUSIONOur data indicate that hypermethylation of hsa-miR-183 is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.

Antibacterial activity of nanostructured Ti-45S5 bioglass-Ag composite against Streptococcus mutans and Staphylococcus aureus

Mechanical alloying and annealing at 1150 °C for 2 h under an argon atmosphere were used to prepare Ti-45S5 bioglass nanocomposites. Ti-45S5 bioglass material was chemically modified by silver. The antibacterial activity of Ti-10% 45S5 bioglass nanocomposite containing silver against Streptococcus mutans and Staphylococcus aureus was studied. Nanocomposites were characterized by X-ray diffraction, scanning electron microscopy equipped with an electron energy dispersive spectrometer and transmission electron microscopy to evaluate phase composition, crystal structure and grain size. In vitro bacterial adhesion study indicated a significantly reduced number of Streptococcus mutans and Staphylococcus aureus on the bulk nanostructured Ti-45S5 bioglass-Ag plate surface in comparison to that on microcrystalline Ti plate surface. Nanostructured Ti-based biomaterials can be considered to be the future generation of dental implants.

Regulation of neuronal survival by DNA methyltransferases

The limited regenerative capacity of neuronal cells requires tight orchestration of cell death and survival regulation in the context of longevity, age-associated diseases as well as during the development of the nervous system. Subordinate to genetic networks epigenetic mechanisms like DNA methylation and histone modifications are involved in the regulation of neuronal development, function and aging. DNA methylation by DNA methyltransferases （DNMTs）, mostly correlated with gene silencing, is a dynamic and reversible process. In addition to their canonical actions performing cytosine methylation, DNMTs influence gene expression by interactions with histone modifying enzymes or complexes increasing the complexity of epigenetic transcriptional networks. DNMTs are expressed in neuronal progenitors, post-mi- totic as well as adult neurons. In this review, we discuss the role and mode of actions of DNMTs including downstream networks in the regulation of neuronal survival in the developing and aging nervous system and its relevance for associated disorders.

Impact of the antiproliferative agent ciclopirox olamine treatment on stem cells proteome

AIM:To investigate the proteome changes of stem cells due to ciclopirox olamine (CPX) treatment compared to control and retinoic acid treated cells. METHODS:Stem cells (SCs) are cells, which have the ability to continuously divide and differentiate into various other kinds of cells. Murine embryonic stem cells (ESCs) and multipotent adult germline stem cells (maG-SCs) were treated with CPX, which has been shown to have an antiproliferative effect on stem cells, and compared to stem cells treated with retinoic acid (RA),which is known to have a differentiating effect on stem cells. Classical proteomic techniques like 2-D gel electrophoresis and differential in-gel electrophoresis (DIGE) were used to generate 2D protein maps from stem cells treated with RA or CPX as well as from non-treated stem cells. The resulting 2D gels were scanned and the digitalized images were collated with the help of Delta 2D software. The differentially expressed proteins were analyzed by a MALDI-TOF-TOF mass spectrometer, and the identified proteins were investigated and categorized using bioinformatics. RESULTS:Treatment of stem cells with CPX, a synthetic antifungal clinically used to treat superficial mycoses, resulted in an antiproliferative effect in vitro, without impairment of pluripotency. To understand the mechanisms induced by CPX treatments which results in arrest of cell cycle without any marked effect on pluripotency, a comparative proteomics study was conducted. The obtained data revealed that the CPX impact on cell proliferation was accompanied with a significant alteration in stem cell proteome. By peptide mass fingerprinting and tandem mass spectrometry combined with searches of protein sequence databases, a set of 316 proteins was identified, corresponding to a library of 125 non-redundant proteins. With proteomic analysis of ESCs and maGSCs treated with CPX and RA, we could identify more than 90 single proteins, which were differently expressed in both cell lines. We could highlight, that CPX treatment of stem cells, with subsequent proliferation inhibition, resulted in an alteration of the expression of 56 proteins compared to nontreated cells, and 54 proteins compared to RA treated cells. Bioinformatics analysis of the regulated proteins demonstrated their involvement in various biological processes. To our interest, a number of proteins have potential roles in the regulation of cell proliferation either directly or indirectly. Furthermore the classification of the altered polypeptides according to their main known/postulated functions revealed that the majority of these proteins are involved in molecular functions like nucleotide binding and metal ion binding, and biological processes like nucleotide biosynthetic processes, gene expression, embryonic development, regulation of transcription, cell cycle processes, RNA and mRNA processing. Proteins, which are involved in nucleotide biosynthetic process and proteolysis, were downregulated in CPX treated cells compared to control, as well as in RA treated cells, which may explain the cell cycle arrest. Moreover, proteins which were involved in cell death, positive regulation of biosynthetic process, response to organic substance, glycolysis, anti-apoptosis, and phosphorylation were downregulated in RA treated cells compared to control and CPX treated cells. CONCLUSION:The CPX treatment of SCs results in downregulation of nucleotide binding proteins and leads to cell cycle stop without impairment of pluripotency.

A simple human cell model for TAU trafficking and tauopathy-related TAU pathology

The microtubule(MT)-associated protein TAU is highly abundant in the axon of human brain neurons,where it binds to and stabilizes MT filaments.Thereby,TAU regulates the dynamic(dis)assembly of MT strands and is involved in a wide range of neuronal functions.In Alzheimer's disease(AD)and other tauopathies,TAU is missorted into the somatodendritic compartment.TAU missorting is accompanied by(or leads to)abnormal TAU phosphorylation,MT destabilization,and loss of dendritic spines and mitochondria,eventually resulting in TAU aggregation,neuronal dysfunction and cell death(Arendt et al.,2016).Strikingly,the mechanisms of TAU sorting,and the detrimental cascade upon its failure,are still not fully understood.

Prevalence of IFNL3 rs4803217 single nucleotide polymorphism and clinical course of chronic hepatitis C

AIM To evaluate the association of IFNL3(IL28B) SNP rs4803217 with severity of disease and treatment outcome in chronic hepatitis C(CHC).METHODS The study enrolled 196 CHC Polish patients(82 women and 114 men in age 20-64) infected with hepatitis C virus(HCV) genotype 1. They were treatment na?ve and qualified to pegylated interferon alpha(PEG-IFN-α) and ribavirin(RBV) therapy. The analyzed baseline parameters included: degree of inflammation, stage of fibrosis, viral load as well as alanine aminotransferase(ALT), asparagine aminotransferase(AST) and total bilirubin(TBIL). The analysis of response to therapy included: sustained virological response(SVR), defined as undetectable serum HCV RNA level six month after completion of 48-wk therapy, and relapse, defined as achieving undetectable viral load at the end of treatment but not SVR. HCV genotyping and HCV RNA quantification were performed using commercially available tests. DNA was isolated from peripheral blood mononuclear cells or from buccal cell swabs. In addition to rs4803217, also single nucleotide polymorphisms(SNPs)(rs12979860, rs8099917 and rs12980275) of known significance in predicting of HCV clearance were analyzed. SNPs were determined by high resolution melt analysis and confirmed by sequencing of amplicons. RESULTS Frequency of rs4803217 genotypes in studied group was as follows: 27.55%; 54.59% and 17.86% for CC, CA and AA, respectively. The rs4803217 SNP, similar to other analyzed SNPs, was not associated with severity of CHC(grade of inflammation, stage of fibrosis, baseline viral load as well as biochemical parameters: ALT, AST, TBIL). It was demonstrated that the rs4803217 C allele is associated with SVR(C vs A: P < 0.0001; dose of C allele: P = 0.0002) and nonrelapse(C vs A: P = 0.001; dose of C allele: P = 0.002). Moreover, it was found that patients with CC genotype have significantly higher response rates as compared with CA/AA patients(P < 0.0001), whereas patients carrying A allele are significantly predisposed to relapse after treatment(P = 0.0007). Moreover, the association of rs4803217 with SVR was comparable to that of rs12979860 and stronger as observed for rs12980275 and rs8099917. Association of rs4803217 with relapse, was the strongest as compared with the other SNPs. The analysis of combined rs4803217 and rs8099917 genotypes demonstrated that additional genotyping of rs8099917 had no significant impact on the prediction of SVR. Multivariate analysis revealed that among analyzed SNPs only rs4803217 is an independent predictor of SVR(P = 0.016) and relapse(P = 0.024). CONCLUSION The rs4803217 SNP is a strong, independent and superior predictor of SVR and relapse in HCV genotype 1 infected CHC patients treated with PEG-IFN-α and RBV.

Spindle Cell Lipoma of the Oral Cavity: A Clinico-Pathologic Analysis of 35 Reported Cases

Spindle-cell lipoma (SCL) is a variant of lipoma, usually presenting in the neck or trunk. SCL of the oral cavity is rare. Some cases of oral SCL have been reported;however, the clinico-pathologic characteristics of oral SCL are still unknown. Here we report on 2 cases and review 33 cases of oral SCL published in the literature. The patients were 23 men and 12 women, with a mean age of 55 years (range 23 - 88 years);there were no children. Tumor sites included: tongue (n = 13), cheek/buccal mucosa (n = 11), floor of mouth (n = 5), lip (n = 2), hard palate (n = 2), alveolar ridge (n = 1), and maxilla (n = 1). The mean tumor size was 20.87 mm (range 3 - 50 mm), smaller than extra-oral lesions. Thirty-three patients (94%) presented with a single lesion that was circumscribed and composed of mature adipocytes admixed with bland spindle cells. The spindle cells were strongly immunoreactive for CD34, whereas the adipocytes were negative. The main differential diagnosis is atypical lipoma/well-differentiated liposarcoma. All lesions underwent local excision and recurrences are rare. Oral SCL do not seem to differ from their extra-oral counterpart, which are much more common and larger in size. SCL should be included in the differential diagnosis of oral cavity mesenchymal tumors.

Is number of chiasmata an etiological factor of male infertility？

Azoospermia and/or severe oligozoospermia can be treated as principal factors of male infertility. Specifically, azoospermia that stands for 1% of males and 10% of infertile male population has been mostly unexplained （idiopathic）, however, it is suspected for its genetic and/or molecular background？ Pertaining to molecular background of severe oligo-astheno-teratozoospermia （OAT） - the meiotic abnormalities are suspected to reach almost 20%.2 Therefore, this potentially addresses a significant number of cases that could find a possible etiological factor. Meiotic abnormalities as the authors point out encompass combination of anomalies in the process of pairing,

Stem cells derived from testis show promise for treating a wide variety of medical conditions

The continuation of the spermatogenic process through-out life relies on a proper regulation of self-renewal and differentiation ofgermline testis stem cells, the spermatogonial stem cells. These are single cells situated on the basal membrane of the seminiferous epithelium. Only 0.03% of all germ cells are spermatogonial stem cells （SSCs） [1-3]. To maintain spermatogenesis, the processes of self-renewal and differentiation of S SCs must be precisely regulated by intrinsic gene expression in the stem cells and extrinsic signals, including soluble factors or adhesion molecules from the surrounding microenvironment, the stem cell niche.

Xenopus Tbx6 mediates posterior patterning via activation of Wnt and FGF signalling

In vertebrates, the patterning of anterior-posterior （AP） axis is a fundamental process during embryogenesis. Wnt and FGF signalling pathways play important roles in regulating the patterning of embryo AP axis. Mouse Tbx6 encodes a transcription factor that has been demonstrated to be involved in the specification of the posterior tissue in mouse embryonic body. Here, we prove that morpholino-induced knockdown of XTbx6 impairs posterior development, indicating the requirement of XTbx6 in this process. Meanwhile, gain of XTbx6 function is sufficient to induce ectopic posterior structures in Xenopus embryos. Furthermore, XTbx6 activates the expression of Xwnt8 and FGF8, which are two mediators of posterior development, suggesting a mechanism by which XTbx6 modulates posterior patterning via Wnt and FGF signalling pathway activation.

Fine-tuning of cortical progenitor proliferation by thalamic afferents

During cerebral cortical cortex neurogenesis two major types of progenitors generate a variety of morphologically and functionally diverse projection neurons destined for the different cortical layers in non-gyrified mice. Radial glia cells （RGCs） undergo mitosis in the cortical ventricular zone and exhibit an apical-basal cell polarity, whereas non-polar intermediate progenitor cells （IPCs） divide basally in the subventricular zone （Franco and Muller, 2013; Taverna et al., 2014）.