GPR34-mediated sensing of lysophosphatidylserine released by apoptotic neutrophils activates type 3 innate lymphoid cells to mediate tissue repair.

Neutrophils migrate rapidly to damaged tissue and play critical roles in host defense and tissue homeostasis. Here we investigated the mechanisms whereby neutrophils participate in tissue repair. In an intestinal epithelia injury model, neutrophil depletion exacerbated colitis and associated with reduced interleukin (IL)-22 and limited activation of type 3 innate lymphoid cells (ILC3s). Co-culture with neutrophils activated ILC3s in a manner dependent on neutrophil apoptosis. Metabolomic analyses revealed that lysophosphatidylserine (LysoPS) from apoptotic neutrophils directly stimulated ILC3 activation. ILC3-specific deletion of Gpr34, encoding the LysoPS receptor GPR34, or inhibition of downstream PI3K-AKT or ERK suppressed IL-22 production in response to apoptotic neutrophils. Gpr34-/- mice exhibited compromised ILC3 activation and tissue repair during colon injury, and neutrophil depletion abrogated these defects. GPR34 deficiency in ILC3s limited IL-22 production and tissue repair in vivo in settings of colon and skin injury. Thus, GPR34 is an ILC3-expressed damage-sensing receptor that triggers tissue repair upon recognition of dying neutrophils.

Human parainfluenza virus 3 vaccine candidates attenuated by codon-pair deoptimization are immunogenic and protective in hamsters.

Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.

E3 ligase RNF2 inhibits porcine circovirus type 3 replication by targeting its capsid protein for ubiquitination-dependent degradation.

Porcine circovirus type 3 (PCV3) is closely associated with various diseases, such as the porcine dermatitis, nephropathy syndrome, and multisystemic clinicopathological diseases. PCV3-associated diseases are increasingly recognized as severe diseases in the global swine industry. Ring finger protein 2 (RNF2), an E3 ubiquitin ligase exclusively located in the nucleus, contributes to various biological processes. This ligase interacts with the PCV3 Cap. However, its role in PCV3 replication remains unclear. This study confirmed that the nuclear localization signal domain of the Cap and the RNF2 N-terminal RING domain facilitate the interaction between the Cap and RNF2. Furthermore, RNF2 promoted the binding of K48-linked polyubiquitination chains to lysine at positions 139 and 140 (K139 and K140) of the PCV3 Cap, thereby degrading the Cap. RNF2 knockdown and overexpression increased or decreased PCV3 replication, respectively. Moreover, the RING domain-deleted RNF2 mutant eliminated the RNF2-induced degradation of the PCV3 Cap and RNF2-mediated inhibition of viral replication. This indicates that both processes were associated with its E3 ligase activity. Our findings demonstrate that RNF2 can interact with and degrade the PCV3 Cap via its N-terminal RING domain in a ubiquitination-dependent manner, thereby inhibiting PCV3 replication.IMPORTANCEPorcine circovirus type 3 is a recently described pathogen that is prevalent worldwide, causing substantial economic losses to the swine industry. However, the mechanisms through which host proteins regulate its replication remain unclear. Here, we demonstrate that ring finger protein 2 inhibits porcine circovirus type 3 replication by interacting with and degrading the Cap of this pathogen in a ubiquitination-dependent manner, requiring its N-terminal RING domain. Ring finger protein 2-mediated degradation of the Cap relies on its E3 ligase activity and the simultaneous existence of K139 and K140 within the Cap. These findings reveal the mechanism by which this protein interacts with and degrades the Cap to inhibit porcine circovirus type 3 replication. This consequently provides novel insights into porcine circovirus type 3 pathogenesis and facilitates the development of preventative measures against this pathogen.

Treatment with sodium butyrate induces autophagy resulting in therapeutic benefits for spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 (SCA3, also known as Machado Joseph disease) is a fatal neurodegenerative disease caused by the expansion of the trinucleotide repeat region within the ATXN3/MJD gene. Mutation of ATXN3 causes formation of ataxin-3 protein aggregates, neurodegeneration, and motor deficits. Here we investigated the therapeutic potential and mechanistic activity of sodium butyrate (SB), the sodium salt of butyric acid, a metabolite naturally produced by gut microbiota, on cultured SH-SY5Y cells and transgenic zebrafish expressing human ataxin-3 containing 84 glutamine (Q) residues to model SCA3. SCA3 SH-SY5Y cells were found to contain high molecular weight ataxin-3 species and detergent-insoluble protein aggregates. Treatment with SB increased the activity of the autophagy protein quality control pathway in the SCA3 cells, decreased the presence of ataxin-3 aggregates and presence of high molecular weight ataxin-3 in an autophagy-dependent manner. Treatment with SB was also beneficial in vivo, improving swimming performance, increasing activity of the autophagy pathway, and decreasing the presence of insoluble ataxin-3 protein species in the transgenic SCA3 zebrafish. Co-treating the SCA3 zebrafish with SB and chloroquine, an autophagy inhibitor, prevented the beneficial effects of SB on zebrafish swimming, indicating that the improved swimming performance was autophagy-dependent. To understand the mechanism by which SB induces autophagy we performed proteomic analysis of protein lysates from the SB-treated and untreated SCA3 SH-SY5Y cells. We found that SB treatment had increased activity of Protein Kinase A and AMPK signaling, with immunoblot analysis confirming that SB treatment had increased levels of AMPK protein and its substrates. Together our findings indicate that treatment with SB can increase activity of the autophagy pathway process and that this has beneficial effects in vitro and in vivo. While our results suggested that this activity may involve activity of a PKA/AMPK-dependent process, this requires further confirmation. We propose that treatment with sodium butyrate warrants further investigation as a potential treatment for neurodegenerative diseases underpinned by mechanisms relating to protein aggregation including SCA3.

Disrupted cerebellar structural connectome in spinocerebellar ataxia type 3 and its association with transcriptional profiles.

Spinocerebellar ataxia type 3 (SCA3) is primarily characterized by progressive cerebellar degeneration, including gray matter atrophy and disrupted anatomical and functional connectivity. The alterations of cerebellar white matter structural network in SCA3 and the underlying neurobiological mechanism remain unknown. Using a cohort of 20 patients with SCA3 and 20 healthy controls, we constructed cerebellar structural networks from diffusion MRI and investigated alterations of topological organization. Then, we mapped the alterations with transcriptome data from the Allen Human Brain Atlas to identify possible biological mechanisms for regional selective vulnerability to white matter damage. Compared with healthy controls, SCA3 patients exhibited reduced global and nodal efficiency, along with a widespread decrease in edge strength, particularly affecting edges connected to hub regions. The strength of inter-module connections was lower in SCA3 group and negatively correlated with the Scale for the Assessment and Rating of Ataxia score, International Cooperative Ataxia Rating Scale score, and cytosine-adenine-guanine repeat number. Moreover, the transcriptome-connectome association study identified the expression of genes involved in synapse-related and metabolic biological processes. These findings suggest a mechanism of white matter vulnerability and a potential image biomarker for the disease severity, providing insights into neurodegeneration and pathogenesis in this disease.

ASOs are an effective treatment for disease-associated oligodendrocyte signatures in premanifest and symptomatic SCA3 mice.

Spinocerebellar ataxia type 3 (SCA3) is the most common dominantly inherited ataxia. Currently, no preventive or disease-modifying treatments exist for this progressive neurodegenerative disorder, although efforts using gene silencing approaches are under clinical trial investigation. The disease is caused by a CAG repeat expansion in the mutant gene, ATXN3, producing an enlarged polyglutamine tract in the mutant protein. Similar to other paradigmatic neurodegenerative diseases, studies evaluating the pathogenic mechanism focus primarily on neuronal implications. Consequently, therapeutic interventions often overlook non-neuronal contributions to disease. Our lab recently reported that oligodendrocytes display some of the earliest and most progressive dysfunction in SCA3 mice. Evidence of disease-associated oligodendrocyte signatures has also been reported in other neurodegenerative diseases, including Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease. Here, we assess the effects of anti-ATXN3 antisense oligonucleotide (ASO) treatment on oligodendrocyte dysfunction in premanifest and symptomatic SCA3 mice. We report a severe, but modifiable, deficit in oligodendrocyte maturation caused by the toxic gain-of-function of mutant ATXN3 early in SCA3 disease that is transcriptionally, biochemically, and functionally rescued with anti-ATXN3 ASO. Our results highlight the promising use of an ASO therapy across neurodegenerative diseases that requires glial targeting in addition to affected neuronal populations.

The deubiquitinase function of ataxin-3 and its role in the pathogenesis of Machado-Joseph disease and other diseases.

Machado-Joseph disease (MJD) is a devastating and incurable neurodegenerative disease characterised by progressive ataxia, difficulty speaking and swallowing. Consequently, affected individuals ultimately become wheelchair dependent, require constant care, and face a shortened life expectancy. The monogenic cause of MJD is expansion of a trinucleotide (CAG) repeat region within the ATXN3 gene, which results in polyglutamine (polyQ) expansion within the resultant ataxin-3 protein. While it is well established that the ataxin-3 protein functions as a deubiquitinating (DUB) enzyme and is therefore critically involved in proteostasis, several unanswered questions remain regarding the impact of polyQ expansion in ataxin-3 on its DUB function. Here we review the current literature surrounding ataxin-3's DUB function, its DUB targets, and what is known regarding the impact of polyQ expansion on ataxin-3's DUB function. We also consider the potential neuroprotective effects of ataxin-3's DUB function, and the intersection of ataxin-3's role as a DUB enzyme and regulator of gene transcription. Ataxin-3 is the principal pathogenic protein in MJD and also appears to be involved in cancer. As aberrant deubiquitination has been linked to both neurodegeneration and cancer, a comprehensive understanding of ataxin-3's DUB function is important for elucidating potential therapeutic targets in these complex conditions. In this review, we aim to consolidate knowledge of ataxin-3 as a DUB and unveil areas for future research to aid therapeutic targeting of ataxin-3's DUB function for the treatment of MJD and other diseases.

A case of convergent evolution: Several viral and bacterial pathogens hijack RSK kinases through a common linear motif.

Microbes have been coevolving with their host for millions of years, exploiting host resources to their own benefit. We show that viral and bacterial pathogens convergently evolved to hijack cellular mitogen-activated protein kinase (MAPK) p90-ribosomal S6-kinases (RSKs). Theiler's virus leader (L) protein binds RSKs and prevents their dephosphorylation, thus maintaining the kinases active. Recruitment of RSKs enables L-protein-mediated inhibition of eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2 or PKR) and stress granule formation. Strikingly, ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) and YopM protein of Yersinia use the same peptide motif as L to recruit and activate RSKs. All three proteins interact with a conserved surface-located loop of RSKs, likely acting as an allosteric regulation site. Some unrelated viruses and bacteria thus evolved to harness RSKs in a common fashion, yet to target distinct aspects of innate immunity. As documented for Varicella zoster virus ORF11, additional pathogens likely evolved to hijack RSKs, using a similar short linear motif.

cUMP elicits inter-endothelial gap formation during Pseudomonas aeruginosa infection.

P. aeruginosa utilizes a type 3 secretion system to intoxicate host cells with the nucleotidyl cyclase ExoY. After activation by its host cell cofactor, filamentous actin, ExoY produces purine and pyrimidine cyclic nucleotides, including cAMP, cGMP, and cUMP. ExoY-generated cyclic nucleotides promote inter-endothelial gap formation, impair motility, and arrest cell growth. The disruptive activities of cAMP and cGMP during P. aeruginosa infection are established; however, little is known about the function of cUMP. Here, we tested the hypothesis that cUMP contributes to endothelial cell barrier disruption during P. aeruginosa infection. Utilizing a membrane permeable cUMP analog, cUMP-AM, we revealed that during infection with catalytically inactive ExoY, cUMP promotes inter-endothelial gap formation in cultured PMVECs and contributes to increased filtration coefficient in the isolated perfused lung. These findings indicate that cUMP contributes to endothelial permeability during P. aeruginosa lung infection.

Compressed cerebellar functional connectome hierarchy in spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 (SCA3) is an inherited movement disorder characterized by a progressive decline in motor coordination. Despite the extensive functional connectivity (FC) alterations reported in previous SCA3 studies in the cerebellum and cerebellar-cerebral pathways, the influence of these FC disturbances on the hierarchical organization of cerebellar functional regions remains unclear. Here, we compared 35 SCA3 patients with 48 age- and sex-matched healthy controls using a combination of voxel-based morphometry and resting-state functional magnetic resonance imaging to investigate whether cerebellar hierarchical organization is altered in SCA3. Utilizing connectome gradients, we identified the gradient axis of cerebellar hierarchical organization, spanning sensorimotor to transmodal (task-unfocused) regions. Compared to healthy controls, SCA3 patients showed a compressed hierarchical organization in the cerebellum at both voxel-level (p < .05, TFCE corrected) and network-level (p < .05, FDR corrected). This pattern was observed in both intra-cerebellar and cerebellar-cerebral gradients. We observed that decreased intra-cerebellar gradient scores in bilateral Crus I/II both negatively correlated with SARA scores (left/right Crus I/II: r = -.48/-.50, p = .04/.04, FDR corrected), while increased cerebellar-cerebral gradients scores in the vermis showed a positive correlation with disease duration (r = .48, p = .04, FDR corrected). Control analyses of cerebellar gray matter atrophy revealed that gradient alterations were associated with cerebellar volume loss. Further FC analysis showed increased functional connectivity in both unimodal and transmodal areas, potentially supporting the disrupted cerebellar functional hierarchy uncovered by the gradients. Our findings provide novel evidence regarding alterations in the cerebellar functional hierarchy in SCA3.

Ubiquitination-dependent degradation of nucleolin mediated by porcine circovirus type 3 capsid protein.

IMPORTANCE: Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes multisystem disease in pigs and poses a severe threat to the swine industry. However, the mechanisms of how PCV3 uses host proteins to regulate its own life cycle are not well understood. In this study, we found that PCV3 capsid protein interacts with nucleolin and degrades it. Degradation of nucleolin by the PCV3 capsid protein requires recruitment of the enzyme RNF34, which is transported to the nucleolus from the cytoplasm in the presence of the PCV3 capsid protein. Nucleolin also decreases PCV3 replication by promoting the release of interferon ß. These findings clarify the mechanism by which nucleolin modulates PCV3 replication in cells, thereby facilitating to provide an important strategy for preventing and controlling PCV3 infection.

Generation of Human Embryonic Stem Cell-Derived Lung Organoids for Modeling Infection and Replication Differences between Human Adenovirus Types 3 and 55 and Evaluating Potential Antiviral Drugs.

Human adenoviruses type 3 (HAdV-3) and type 55 (HAdV-55) are frequently encountered, highly contagious respiratory pathogens with high morbidity rate. In contrast to HAdV-3, one of the most predominant types in children, HAdV-55 is a reemergent pathogen associated with more severe community-acquired pneumonia (CAP) in adults, especially in military camps. However, the infectivity and pathogenicity differences between these viruses remain unknown as in vivo models are not available. Here, we report a novel system utilizing human embryonic stem cells-derived 3-dimensional airway organoids (hAWOs) and alveolar organoids (hALOs) to investigate these two viruses. Firstly, HAdV-55 replicated more robustly than HAdV-3. Secondly, cell tropism analysis in hAWOs and hALOs by immunofluorescence staining revealed that HAdV-55 infected more airway and alveolar stem cells (basal and AT2 cells) than HAdV-3, which may lead to impairment of self-renewal functions post-injury and the loss of cell differentiation in lungs. Additionally, the viral life cycles of HAdV-3 and -55 in organoids were also observed using Transmission Electron Microscopy. This study presents a useful pair of lung organoids for modeling infection and replication differences between respiratory pathogens, illustrating that HAdV-55 has relatively higher replication efficiency and more specific cell tropism in human lung organoids than HAdV-3, which may result in relatively higher pathogenicity and virulence of HAdV-55 in human lungs. The model system is also suitable for evaluating potential antiviral drugs, as demonstrated with cidofovir. IMPORTANCE Human adenovirus (HAdV) infections are a major threat worldwide. HAdV-3 is one of the most predominant respiratory pathogen types found in children. Many clinical studies have reported that HAdV-3 causes less severe disease. In contrast, HAdV-55, a reemergent acute respiratory disease pathogen, is associated with severe community-acquired pneumonia in adults. Currently, no ideal in vivo models are available for studying HAdVs. Therefore, the mechanism of infectivity and pathogenicity differences between human adenoviruses remain unknown. In this study, a useful pair of 3-dimensional (3D) airway organoids (hAWOs) and alveolar organoids (hALOs) were developed to serve as a model. The life cycles of HAdV-3 and HAdV-55 in these human lung organoids were documented for the first time. These 3D organoids harbor different cell types, which are similar to the ones found in humans. This allows for the study of the natural target cells for infection. The finding of differences in replication efficiency and cell tropism between HAdV-55 and -3 may provide insights into the mechanism of clinical pathogenicity differences between these two important HAdV types. Additionally, this study provides a viable and effective in vitro tool for evaluating potential anti-adenoviral treatments.

Hematopoietic stem cell transplantation or enzyme replacement therapy in Gaucher disease type 3.

Gaucher disease (GD) is a lysosomal storage disorder with glucocerebroside accumulation in the macrophages. The disease is divided into three types based on neurocognitive involvement with GD1 having no involvement while the acute (GD2) and chronic (GD3) are neuronopathic. The non-neurological symptoms of GD3 are well treated with enzyme replacement therapy (ERT) which has replaced hematopoietic stem cell transplantation (HSCT). ERT is unable to prevent neurological progression as the enzyme cannot cross the blood-brain barrier. In this retrospective study, we report the general, neurocognitive, and biochemical outcomes of three siblings with GD3 after treatment with ERT or HSCT. Two were treated with HSCT (named HSCT1 and HSCT2) and one with ERT (ERT1). All patients were homozygous for the c.1448 T > C, (p.Leu483Pro) variant in the GBA1 gene associated with GD3. ERT1 experienced neurocognitive progression with development of seizures, oculomotor apraxia, perceptive hearing loss and mental retardation. HSCT1 had no neurological manifestations, while HSCT2 developed perceptive hearing loss and low IQ. Chitotriosidase concentrations were normal in plasma and cerebrospinal fluid (CSF) for HSCT1 and HSCT2, but both were markedly elevated in ERT1. We report a better neurological outcome and a normalization of chitotriosidase in the two siblings treated with HSCT compared to the ERT-treated sibling. With the advancements in HSCT over the past 25 years, we may reconsider using HSCT in GD3 to achieve a better neurological outcome and limit disease progression.

The parkin V380L variant is a genetic modifier of Machado-Joseph disease with impact on mitophagy.

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative spinocerebellar ataxia caused by a polyglutamine-coding CAG repeat expansion in the ATXN3 gene. While the CAG length correlates negatively with the age at onset, it accounts for approximately 50% of its variability only. Despite larger efforts in identifying contributing genetic factors, candidate genes with a robust and plausible impact on the molecular pathogenesis of MJD are scarce. Therefore, we analysed missense single nucleotide polymorphism variants in the PRKN gene encoding the Parkinson's disease-associated E3 ubiquitin ligase parkin, which is a well-described interaction partner of the MJD protein ataxin-3, a deubiquitinase. By performing a correlation analysis in the to-date largest MJD cohort of more than 900 individuals, we identified the V380L variant as a relevant factor, decreasing the age at onset by 3 years in homozygous carriers. Functional analysis in an MJD cell model demonstrated that parkin V380L did not modulate soluble or aggregate levels of ataxin-3 but reduced the interaction of the two proteins. Moreover, the presence of parkin V380L interfered with the execution of mitophagy-the autophagic removal of surplus or damaged mitochondria-thereby compromising cell viability. In summary, we identified the V380L variant in parkin as a genetic modifier of MJD, with negative repercussions on its molecular pathogenesis and disease age at onset.

Generation of human ILC3 from allogeneic and autologous CD34+ hematopoietic progenitors toward adoptive transfer.

Type 3 innate lymphoid cells (ILC3) are important in tissue homeostasis. In the gut, ILC3 repair damaged epithelium and suppress inflammation. In allogeneic hematopoietic cell transplantation (HCT), ILC3 protect against graft-versus-host disease (GvHD), most likely by restoring tissue damage and preventing inflammation. We hypothesize that supplementing HCT grafts with interleukin-22 (IL-22)-producing ILC3 may prevent acute GvHD. We therefore explored ex vivo generation of human IL-22-producing ILC3 from hematopoietic stem and progenitor cells (HSPC) obtained from adult, neonatal and fetal sources. We established a stroma-free system culturing human cord blood-derived CD34+ HSPC with successive cytokine mixes for 5 weeks. We analyzed the presence of phenotypically defined ILC, their viability, proliferation and IL-22 production (after stimulation) by flow cytometry and enzyme-linked immunosorbent assay (ELISA). We found that the addition of recombinant human IL-15 and the enhancer of zeste homolog 1/2 inhibitor UNC1999 promoted ILC3 generation. Similar results were demonstrated when UNC1999 was added to CD34+ HSPC derived from healthy adult granulocyte colony-stimulating factor mobilized peripheral blood and bone marrow, but not fetal liver. UNC1999 did not negatively impact IL-22 production in any of the HSPC sources. Finally, we observed that autologous HSPC mobilized from the blood of adults with hematological malignancies also developed into ILC3, albeit with a significantly lower capacity. Together, we developed a stroma-free protocol to generate large quantities of IL-22-producing ILC3 from healthy adult human HSPC that can be applied for adoptive transfer to prevent GvHD after allogeneic HCT.

Genetic Epidemiology and Clinical Characteristics of Patients with Spinocerebellar Ataxias in an Unexplored Brazilian State, Using Strategies for Resource-Limited Settings.

Spinocerebellar ataxias (SCAs) have a worldwide average prevalence of 2.7 cases per 100,000 individuals, with significant geographic variability. This study aimed to develop resource-limited strategies to detect and characterize the frequency and genetic-clinical profile of SCAs in an unexplored population from Alagoas State, a low Human Development Index state in northeastern Brazil. Active search strategies were employed to identify individuals with a diagnosis or clinical suspicion of SCAs, and a protocol for clinical and molecular evaluation was applied in collaboration with a reference center in Neurogenetics. A total of 73 individuals with SCAs were identified, with a minimum estimated prevalence of 2.17 cases per 100,000 inhabitants. SCA3 was the most common type (75.3%), followed by SCA7 (15.1%), SCA1 (6.8%), and SCA2 (2.7%). Patients with SCA3 subphenotype 2 were the most predominant. Detailed analysis of patients with SCA3 and SCA7 revealed age at onset and clinical features congruent with other studies, with gait disturbance and reduced visual capacity in SCA7 as the main initial manifestations. The study also identified many asymptomatic individuals at risk of developing SCAs. These findings demonstrate that simple and collaborative strategies can enhance the detection capacity of rare diseases such as SCAs in resource-limited settings and that Alagoas State has a minimum estimated prevalence of SCAs similar to the world average.

Caffeine Consumption and Interaction with ADORA2A, CYP1A2 and NOS1 Variants Do Not Influence Age at Onset of Machado-Joseph Disease.

BACKGROUND: The age at onset (AO) of Machado-Joseph disease (SCA3/MJD), a disorder due to an expanded CAG repeat (CAGexp) in ATXN3, is quite variable and the role of environmental factors is still unknown. Caffeine was associated with protective effects against other neurodegenerative diseases, and against SCA3/MJD in transgenic mouse models. We aimed to evaluate whether caffeine consumption and its interaction with variants of caffeine signaling/metabolization genes impact the AO of this disease. METHODS: a questionnaire on caffeine consumption was applied to adult patients and unrelated controls living in Rio Grande do Sul, Brazil. AO and CAGexp were previously determined. SNPs rs5751876 (ADORA2A), rs2298383 (ADORA2A), rs762551 (CYP1A2) and rs478597 (NOS1) were genotyped. AO of subgroups were compared, adjusting the CAGexp to 75 repeats (p < 0.05). RESULTS: 171/179 cases and 98/100 controls consumed caffeine. Cases with high and low caffeine consumption (more or less than 314.5 mg of caffeine/day) had mean (SD) AO of 35.05 (11.44) and 35.43 (10.08) years (p = 0.40). The mean (SD) AO of the subgroups produced by the presence or absence of caffeine-enhancing alleles in ADORA2A (T allele at rs5751876 and rs2298383), CYP1A2 (C allele) and NOS1 (C allele) were all similar (p between 0.069 and 0.516). DISCUSSION: Caffeine consumption was not related to changes in the AO of SCA3/MJD, either alone or in interaction with protective genotypes at ADORA2A, CYP1A2 and NOS1.

Effect of Regional Brain Activity Following Repeat Transcranial Magnetic Stimulation in SCA3: A Secondary Analysis of a Randomized Clinical Trial.

Repetitive transcranial magnetic stimulation (rTMS), a noninvasive neuroregulatory technique used to treat neurodegenerative diseases, holds promise for spinocerebellar ataxia type 3 (SCA3) treatment, although its efficacy and mechanisms remain unclear. This study aims to observe the short-term impact of cerebellar rTMS on motor function in SCA3 patients and utilize resting-state functional magnetic resonance imaging (RS-fMRI) to assess potential therapeutic mechanisms. Twenty-two SCA3 patients were randomly assigned to receive actual rTMS (AC group, n = 11, three men and eight women; age 32-55 years) or sham rTMS (SH group, n = 11, three men and eight women; age 26-58 years). Both groups underwent cerebellar rTMS or sham rTMS daily for 15 days. The primary outcome measured was the ICARS scores and parameters for regional brain activity. Compared to baseline, ICARS scores decreased more significantly in the AC group than in the SH group after the 15-day intervention. Imaging indicators revealed increased Amplitude of Low Frequency Fluctuation (ALFF) values in the posterior cerebellar lobe and cerebellar tonsil following AC stimulation. This study suggests that rTMS enhances motor functions in SCA3 patients by modulating the excitability of specific brain regions and associated pathways, reinforcing the potential clinical utility of rTMS in SCA3 treatment. The Chinese Clinical Trial Registry identifier is ChiCTR1800020133.

Serum PRO-C3 is useful for risk prediction and fibrosis assessment in MAFLD with chronic kidney disease in an Asian cohort.

BACKGROUND: Metabolic dysfunction-associated fatty liver disease (MAFLD) is an emerging risk factor for chronic kidney disease (CKD). N-terminal propeptide of collagen type 3 (PRO-C3) is a biomarker of advanced fibrosis in MAFLD and PRO-C3 may be involved in renal fibrosis. We aimed to use PRO-C3 measurements to generate a new algorithmic score to test the prediction of MAFLD with chronic kidney disease (MAFLD-CKD). METHODS: A derivation and independent validation cohort of 750 and 129 Asian patients with biopsy-confirmed MAFLD were included. Serum PRO-C3 concentration was measured and regression analyses were performed to examine associations with MAFLD-CKD. A derivative algorithm for MAFLD-CKD risk prediction was evaluated with receiver operator characteristic (ROC) curve analysis. RESULTS: The study included two Asian cohorts (n = 180 with MAFLD-CKD; mean-eGFR: 94.93 mL/min/1.73 m2; median-urinary albumin-to-creatinine ratio: 6.58 mg/mmol). PRO-C3 was associated with the severity of MAFLD-CKD and independently associated with MAFLD-CKD (adjusted odds ratio = 1.16, 95% confidence interval [CI]: 1.08-1.23, p < .001). A new non-invasive score (termed PERIOD) including PRO-C3 efficiently predicted MAFLD-CKD (AUROC = .842, 95% CI: .805-.875). Accuracy, specificity and negative predictive values were 80.2%, 85.1% and 88.4%, respectively. In the validation cohort, the PERIOD score had good diagnostic performance (AUROC = .807, 95% CI: .691-.893) with similar results in all patient subgroups. In the MAFLD-CKD subgroup, the accuracy for identifying advanced fibrosis was further improved by combining the PRO-C3-based ADAPT with the Agile 3+ scores (AUROC = .90, 95% CI: .836-.964). CONCLUSIONS: The PERIOD score is helpful for accurately predicting the risk of MAFLD-CKD. PRO-C3 can also be used to assess liver fibrosis in people with MAFLD-CKD.

Type 3 resistant starch from Canna edulis reduce lipid levels in patients with mild hyperlipidemia through altering gut microbiome: A double- blind randomized controlled trial.

Type 3 resistant starch from Canna edulis (Ce-RS3) is an insoluble dietary fiber which could improve blood lipids in animals, but clinically robust evidence is still lacking. We performed a double-blind randomized controlled trial to assess the effects of Ce-RS3 on lipids in mild hyperlipidemia. One hundred and fifteen patients were included followed the recruitment criteria, and were randomly allocated to receive Ce-RS3 or placebo (native starch from Canna edulis) for 12 weeks (20 g/day). In addition to serum lipids, complete blood counts, serum inflammatory factors, antioxidant indexes, and dietary survey, 16 S rRNA sequencing technique was utilized to analyze the gut microbiota alterations. Targeted quantitative metabolomics (TQM) was used to detect metabolite changes. Compared with the placebo, Ce- RS3 significantly decreased levels of total cholesterol, lowdensity lipoprotein cholesterol, and non-high-density lipoprotein cholesterol, and increased the glutathione peroxidase. Based on the 16 S rRNA sequencing, TQM, the correlation analysis, as well as the Kyoto Encyclopedia of Genes (KEGG) and Genomes and Human Metabolome Database (HMDB) analysis, we found that Ce-RS3 could increase the abundances of genera Faecalibacterium and Agathobacter, while reduce the abundances of genera norank_f_Ruminococcaceae and Christensenellaceae_R-7_ group to regulate phenylalanine metabolism, which could reduce the fatty acid biosynthesis and fatty acid elongation in the mitochondria to lower blood lipids. Conclusively, we firstly confirmed the feasibility of Ce-RS3 for clinical application, which presents a novel, effective therapy for the mild hyperlipidemia. (Chictr. org. cn. Clinical study on anti-mild hyperlipidemia of Canna edulis RS3 resistant starch, ID Number: ChiCTR2200062871).