Novel Scorpion Toxin ω-Buthitoxin-Hf1a Selectively Inhibits Calcium Influx via CaV3.3 and CaV3.2 and Alleviates Allodynia in a Mouse Model of Acute Postsurgical Pain.

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 µM) and CaV3.3 (IC50 = 0.49 µM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.

Targeting the tumor microenvironment for treating double refractory chronic lymphocytic leukemia.

The introduction of BTK inhibitors and BCL2 antagonists to the treatment of chronic lymphocytic leukemia has revolutionized therapy and improved patient outcomes. These agents have replaced chemoimmunotherapy as standard of care. Despite this progress, a new group of patients is currently emerging, which has become refractory or intolerant to both classes of agents, creating an unmet medical need. Here, we propose that the targeted modulation of the tumor microenvironment provides new therapeutic options for this group of "double refractory" patients. Furthermore, we outline a sequential strategy for tumor microenvironment-directed combination therapies in CLL that can be tested in clinical protocols.

Case Report: Sudden very late-onset near fatal PD1 inhibitor-associated myocarditis with out-of-hospital cardiac arrest after >2.5 years of pembrolizumab treatment.

Introduction: Immune checkpoint inhibitors have advanced the outcomes of many different types of cancer. A rare but extraordinarily severe complication of these agents resembles immune checkpoint inhibitor-related myocarditis, which typically occurs within the first few weeks after treatment initiation with a mortality of 25%-50%. Case report: A 57-year-old woman had uneventfully received pembrolizumab for metastatic non-small cell lung cancer for over 2.5 years and was admitted after an out-of-hospital cardiac arrest due to ventricular fibrillation. After successful cardiopulmonary resuscitation, the initial diagnostic work-up showed elevated cardiac enzymes and a limited left-ventricular ejection fraction, while coronary angiography did not show relevant stenosis. Despite cardiac MRI being unsuggestive of myocarditis, myocardial biopsies were obtained and histologically confirmed anti-PD-1 antibody-associated myocarditis. After the initiation of prednisone at 1 mg/kg body weight, the patient gradually recovered and was discharged three weeks later with markedly improved cardiac function. Conclusion: This case resembles the first description of a very late onset irMyocarditis, occurring over 2.5 years after the start of treatment. It demonstrates the importance of contemplating that severe immune-related toxicities with a sudden onset clinical presentation may occur even after long uneventful periods of anti-PD-1 immune checkpoint inhibitor treatment. Furthermore, it underlines the critical importance of myocardial biopsies in this setting, especially when cardiac MRI remains inconclusive. Moreover, it demonstrates the necessity and benefits of early immunosuppressive treatment if immune-related myocarditis is considered a differential diagnosis.

A syndromic neurodevelopmental disorder caused by rare variants in PPFIA3.

PPFIA3 encodes the protein-tyrosine phosphatase, receptor-type, F-polypeptide-interacting-protein-alpha-3 (PPFIA3), which is a member of the LAR-protein-tyrosine phosphatase-interacting-protein (liprin) family involved in synapse formation and function, synaptic vesicle transport, and presynaptic active zone assembly. The protein structure and function are evolutionarily well conserved, but human diseases related to PPFIA3 dysfunction are not yet reported in OMIM. Here, we report 20 individuals with rare PPFIA3 variants (19 heterozygous and 1 compound heterozygous) presenting with developmental delay, intellectual disability, hypotonia, dysmorphisms, microcephaly or macrocephaly, autistic features, and epilepsy with reduced penetrance. Seventeen unique PPFIA3 variants were detected in 18 families. To determine the pathogenicity of PPFIA3 variants in vivo, we generated transgenic fruit flies producing either human wild-type (WT) PPFIA3 or five missense variants using GAL4-UAS targeted gene expression systems. In the fly overexpression assays, we found that the PPFIA3 variants in the region encoding the N-terminal coiled-coil domain exhibited stronger phenotypes compared to those affecting the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin-α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 function is partially conserved in the fly. However, two of the tested variants failed to rescue the lethality at the larval stage and one variant failed to rescue lethality at the adult stage. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant-negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Sleep apnea multi-level surgery trial: long-term observational outcomes.

STUDY OBJECTIVES: The sleep apnea multi-level surgery (SAMS) randomized clinical trial showed surgery improved outcomes at 6 months compared to ongoing medical management in patients with moderate or severe obstructive sleep apnea (OSA) who failed continuous positive airway pressure therapy. This study reports the long-term outcomes of the multi-level surgery as a case series. METHODS: Surgical participants were reassessed >2 years postoperatively with the same outcomes reported in the main SAMS trial. Primary outcomes were apnea-hypopnea index (AHI) and Epworth sleepiness scale (ESS), with secondary outcomes including other polysomnography measures, symptoms, quality of life, and adverse events. Long-term effectiveness (baseline to long-term follow-up [LTFU]) and interval changes (6 month to LTFU) were assessed using mixed effects regression models. Control participants were also reassessed for rate of subsequent surgery and outcomes. RESULTS: 36/48 (75%) of surgical participants were reevaluated (mean (standard deviation)) 3.5 (1.0) years following surgery, with 29 undergoing polysomnography. AHI was 41/h (23) at preoperative baseline and 21/h (18) at follow-up, representing persistent improvement of -24/h (95% CI -32, -17; p < 0.001). ESS was 12.3 (3.5) at baseline and 5.5 (3.9) at follow-up, representing persistent improvement of -6.8 (95% CI -8.3, -5.4; p < 0.001). Secondary outcomes were improved long term, and adverse events were minor. Interval change analysis suggests stability of outcomes. 36/43 (84%) of the control participants were reevaluated, with 25 (69%) reporting subsequent surgery, with symptom and quality of life improvements. CONCLUSION: Multi-level upper airway surgery improves OSA burden with long-term maintenance of treatment effect in adults with moderate or severe OSA in whom conventional therapy failed. CLINICAL TRIAL: Multi-level airway surgery in patients with moderate-severe obstructive sleep apnea (OSA) who have failed medical management to assess change in OSA events and daytime sleepiness; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=366019&isReview=true; ACTRN12614000338662.

Entirely noninvasive outcome prediction in central nervous system lymphomas using circulating tumor DNA.

ABSTRACT: State-of-the-art response assessment of central nervous system lymphoma (CNSL) by magnetic resonance imaging is challenging and an insufficient predictor of treatment outcomes. Accordingly, the development of novel risk stratification strategies in CNSL is a high unmet medical need. We applied ultrasensitive circulating tumor DNA (ctDNA) sequencing to 146 plasma and cerebrospinal fluid (CSF) samples from 67 patients, aiming to develop an entirely noninvasive dynamic risk model considering clinical and molecular features of CNSL. Our ultrasensitive method allowed for the detection of CNSL-derived mutations in plasma ctDNA with high concordance to CSF and tumor tissue. Undetectable plasma ctDNA at baseline was associated with favorable outcomes. We tracked tumor-specific mutations in plasma-derived ctDNA over time and developed a novel CNSL biomarker based on this information: peripheral residual disease (PRD). Persistence of PRD after treatment was highly predictive of relapse. Integrating established baseline clinical risk factors with assessment of radiographic response and PRD during treatment resulted in the development and independent validation of a novel tool for risk stratification: molecular prognostic index for CNSL (MOP-C). MOP-C proved to be highly predictive of outcomes in patients with CNSL (failure-free survival hazard ratio per risk group of 6.60; 95% confidence interval, 3.12-13.97; P < .0001) and is publicly available at www.mop-c.com. Our results highlight the role of ctDNA sequencing in CNSL. MOP-C has the potential to improve the current standard of clinical risk stratification and radiographic response assessment in patients with CNSL, ultimately paving the way toward individualized treatment.

Identification of immune-related candidate biomarkers in plasma of patients with sporadic vestibular schwannoma.

Vestibular schwannoma (VS) is an intracranial tumor arising from neoplastic Schwann cells and typically presenting with hearing loss. The traditional belief that hearing deficit is caused by physical expansion of the VS, compressing the auditory nerve, does not explain the common clinical finding that patients with small tumors can have profound hearing loss, suggesting that tumor-secreted factors could influence hearing ability in VS patients. We conducted profiling of patients' plasma for 66 immune-related factors in patients with sporadic VS (N > 170) and identified and validated candidate biomarkers associated with tumor size (S100B) and hearing (MCP-3). We further identified a nine-biomarker panel (TNR-R2, MIF, CD30, MCP-3, IL-2R, BLC, TWEAK, eotaxin, and S100B) with outstanding discriminatory ability for VS. These findings revealed possible therapeutic targets for VS, providing a unique diagnostic tool that may predict hearing change and tumor growth in VS patients, and may inform the timing of tumor resection to preserve hearing.

Development of a Selective Peptide κ-Opioid Receptor Antagonist by Late-Stage Functionalization with Cysteine Staples.

The κ-opioid receptor (KOR) is an attractive target for the development of novel drugs. KOR agonists are potentially safer pain medications, whereas KOR antagonists are promising drug candidates for the treatment of neuropsychiatric disorders. Hitherto, the vast majority of selective drug leads that have been developed for KOR are small molecules. In this study, novel peptide probes were designed by using an endogenous dynorphin A1-13 sequence as a template for peptide stapling via late-stage cysteine functionalization. Leveraging this strategy, we developed a stable and potent KOR antagonist, CSD-CH2(1,8)-NH2, with approximately 1000-fold improved selectivity for KOR over µ- and δ-opioid receptors. Its potent competitive KOR antagonism was verified in KOR-expressing cells, peripheral dorsal root ganglion neurons, and using the tail-flick and rotarod tests in mice. This work highlights the value of cysteine stapling to develop selective peptide probes to modulate central KOR function, as innovative peptide drug candidates for the treatment of KOR-related illnesses.

Rare variants in PPFIA3 cause delayed development, intellectual disability, autism, and epilepsy.

PPFIA3 encodes the Protein-Tyrosine Phosphatase, Receptor-Type, F Polypeptide-Interacting Protein Alpha-3 (PPFIA3), which is a member of the LAR protein-tyrosine phosphatase-interacting protein (liprin) family involved in synaptic vesicle transport and presynaptic active zone assembly. The protein structure and function are well conserved in both invertebrates and vertebrates, but human diseases related to PPFIA3 dysfunction are not yet known. Here, we report 14 individuals with rare mono-allelic PPFIA3 variants presenting with features including developmental delay, intellectual disability, hypotonia, autism, and epilepsy. To determine the pathogenicity of PPFIA3 variants in vivo , we generated transgenic fruit flies expressing either human PPFIA3 wildtype (WT) or variant protein using GAL4-UAS targeted gene expression systems. Ubiquitous expression with Actin-GAL4 showed that the PPFIA3 variants had variable penetrance of pupal lethality, eclosion defects, and anatomical leg defects. Neuronal expression with elav-GAL4 showed that the PPFIA3 variants had seizure-like behaviors, motor defects, and bouton loss at the 3 rd instar larval neuromuscular junction (NMJ). Altogether, in the fly overexpression assays, we found that the PPFIA3 variants in the N-terminal coiled coil domain exhibited stronger phenotypes compared to those in the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin- α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 protein function is partially conserved in the fly. However, the PPFIA3 variants failed to rescue lethality. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

FOXI3 pathogenic variants cause one form of craniofacial microsomia.

Craniofacial microsomia (CFM; also known as Goldenhar syndrome), is a craniofacial developmental disorder of variable expressivity and severity with a recognizable set of abnormalities. These birth defects are associated with structures derived from the first and second pharyngeal arches, can occur unilaterally and include ear dysplasia, microtia, preauricular tags and pits, facial asymmetry and other malformations. The inheritance pattern is controversial, and the molecular etiology of this syndrome is largely unknown. A total of 670 patients belonging to unrelated pedigrees with European and Chinese ancestry with CFM, are investigated. We identify 18 likely pathogenic variants in 21 probands (3.1%) in FOXI3. Biochemical experiments on transcriptional activity and subcellular localization of the likely pathogenic FOXI3 variants, and knock-in mouse studies strongly support the involvement of FOXI3 in CFM. Our findings indicate autosomal dominant inheritance with reduced penetrance, and/or autosomal recessive inheritance. The phenotypic expression of the FOXI3 variants is variable. The penetrance of the likely pathogenic variants in the seemingly dominant form is reduced, since a considerable number of such variants in affected individuals were inherited from non-affected parents. Here we provide suggestive evidence that common variation in the FOXI3 allele in trans with the pathogenic variant could modify the phenotypic severity and accounts for the incomplete penetrance.

Vestibular dysfunction in neurofibromatosis type 2-related schwannomatosis.

Neurofibromatosis type 2-related schwannomatosis is a genetic disorder characterized by neurologic tumours, most typically vestibular schwannomas that originate on the vestibulo-cochlear nerve(s). Although vestibular symptoms can be disabling, vestibular function has never been carefully analysed in neurofibromatosis type 2-related schwannomatosis. Furthermore, chemotherapy (e.g. bevacizumab) can reduce tumour volume and improve hearing in neurofibromatosis type 2-related schwannomatosis, but nothing is known about its vestibular effects. In this report, we studied the three primary vestibular-mediated behaviours (eye movements, motion perception and balance), clinical vestibular disability (dizziness and ataxia), and imaging and hearing in eight untreated patients with neurofibromatosis type 2-related schwannomatosis and compared their results with normal subjects and patients with sporadic, unilateral vestibular schwannoma tumours. We also examined how bevacizumab affected two patients with neurofibromatosis type 2-related schwannomatosis. Vestibular schwannomas in neurofibromatosis type 2-related schwannomatosis degraded vestibular precision (inverse of variability, reflecting a reduced central signal-to-noise ratio) but not vestibular accuracy (amplitude relative to ideal amplitude, reflecting the central signal magnitude) and caused clinical disability. Bevacizumab improved vestibular precision and clinical disability in both patients with neurofibromatosis type 2-related schwannomatosis but did not affect vestibular accuracy. These results demonstrate that vestibular schwannoma tumours in our neurofibromatosis type 2-related schwannomatosis population degrade the central vestibular signal-to-noise ratio, while bevacizumab improves the signal-to-noise ratio, changes that can be explained mechanistically by the addition (schwannoma) and suppression (bevacizumab) of afferent neural noise.

Neuropathy due to bi-allelic SH3TC2 variants: genotype-phenotype correlation and natural history.

Recessive SH3TC2 variants cause Charcot-Marie-Tooth disease type 4C (CMT4C). CMT4C is typically a sensorimotor demyelinating polyneuropathy, marked by early onset spinal deformities, but its clinical characteristics and severity are quite variable. Clear relationships between pathogenic variants and the spectrum of disease manifestations are to date lacking. Gene replacement therapy has been shown to ameliorate the phenotype in a mouse model of CMT4C, emphasizing the need for natural history studies to inform clinical trial readiness. Data, including both genetic information and clinical characteristics, were compiled from the longitudinal, prospective dataset of the Inherited Neuropathy Consortium, a member of the Rare Diseases Clinical Research Network (INC-RDCRN). The Charcot Marie Tooth Neuropathy Score (CMTNS), Examination Score (CMTES) and the Rasch-weighted CMTES (CMTES-R) were used to describe symptoms, neurological examinations and neurophysiological characteristics. Standardized response means were calculated at yearly intervals and a mixed model for repeated measures was used to estimate the change in CMTES and CMTES-R over time. Fifty-six individuals (59% female), median age 27 years (range 2-67 years) with homozygous or compound heterozygous variants in SH3TC2 were identified, including 34 unique variants, 14 of which have not previously been published. Twenty-eight participants had longitudinal data available. While there was no significant difference in the CMTES in those with protein truncating versus non-protein truncating variants, there were significant differences in the mean ulnar nerve compound muscle action potential amplitude, the mean radial sensory nerve action potential amplitude, and in the prevalence of scoliosis, suggesting the possibility of a milder phenotype in individuals with one or two non-protein-truncating variants. Overall, the mean value of the CMTES was 13, reflecting moderate clinical severity. There was a high rate of scoliosis (81%), scoliosis surgery (36%), and walking difficulty (94%) among study participants. The CMTES and CMTES-R appeared moderately responsive to change over extended follow-up, demonstrating a standardized response mean of 0.81 standard deviation units or 0.71 standard deviation units, respectively, over 3 years. Our analysis represents the largest cross-sectional and only longitudinal study to date, of the clinical phenotype of both adults and children with CMT4C. With the promise of upcoming genetic treatments, these data will further define the natural history of the disease and inform study design in preparation for clinical trials.

Identification of Immune-Related Candidate Biomarkers in Plasma of Patients with Sporadic Vestibular Schwannoma: Candidate Plasma Biomarkers in Vestibular Schwannoma.

Vestibular schwannoma (VS) is intracranial tumor arising from neoplastic Schwann cells, causing hearing loss in about 95% of patients. The traditional belief that hearing deficit is caused by physical expansion of the VS, compressing the auditory nerve, does not explain the common clinical finding that patients with small tumors can have profound hearing loss, suggesting that tumor-secreted factors could influence hearing ability in VS patients. Here, we conducted profiling of patients' plasma for 67 immune-related factors on a large cohort of VS patients (N>120) and identified candidate biomarkers associated with tumor growth (IL-16 and S100B) and hearing (MDC). We identified the 7-biomarker panel composed of MCP-3, BLC, S100B, FGF-2, MMP-14, eotaxin, and TWEAK that showed outstanding discriminatory ability for VS. These findings revealed possible therapeutic targets for VS-induced hearing loss and provided a unique diagnostic tool that may predict hearing change and tumor growth in VS patients and may help inform the ideal timing of tumor resection to preserve hearing.

Expansion of the clinical and molecular spectrum of WWOX-related epileptic encephalopathy.

WWOX biallelic loss-of-function pathogenic single nucleotide variants (SNVs) and copy number variants (CNVs) including exonic deletions and duplications cause WWOX-related epileptic encephalopathy (WOREE) syndrome. This disorder is characterized by refractory epilepsy, axial hypotonia, peripheral hypertonia, progressive microcephaly, and premature death. Here we report five patients with WWOX biallelic predicted null variants identified by exome sequencing (ES), genome sequencing (GS), and/or chromosomal microarray analysis (CMA). SNVs and intragenic deletions of one or more exons were commonly reported in WOREE syndrome patients which made the genetic diagnosis challenging and required a combination of different diagnostic technologies. These patients presented with severe, developmental and epileptic encephalopathy (DEE), and other cardinal features consistent with WOREE syndrome. This report expands the clinical phenotype associated with this condition, including failure to thrive in most patients and epilepsy that responded to a ketogenic diet in three patients. Dysmorphic features and abnormal prenatal findings were not commonly observed. Additionally, recurrent pancreatitis and sensorineural hearing loss each were observed in single patients. In summary, these phenotypic features broaden the clinical spectrum of WOREE syndrome.

The T-type calcium channel Ca V 3.2 regulates bladder afferent responses to mechanical stimuli.

ABSTRACT: The bladder wall is innervated by a complex network of afferent nerves that detect bladder stretch during filling. Sensory signals, generated in response to distension, are relayed to the spinal cord and brain to evoke physiological and painful sensations and regulate urine storage and voiding. Hyperexcitability of these sensory pathways is a key component in the development of chronic bladder hypersensitivity disorders including interstitial cystitis/bladder pain syndrome and overactive bladder syndrome. Despite this, the full array of ion channels that regulate bladder afferent responses to mechanical stimuli have yet to be determined. Here, we investigated the role of low-voltage-activated T-type calcium (Ca V 3) channels in regulating bladder afferent responses to distension. Using single-cell reverse-transcription polymerase chain reaction and immunofluorescence, we revealed ubiquitous expression of Ca V 3.2, but not Ca V 3.1 or Ca V 3.3, in individual bladder-innervating dorsal root ganglia neurons. Pharmacological inhibition of Ca V 3.2 with TTA-A2 and ABT-639, selective blockers of T-type calcium channels, dose-dependently attenuated ex-vivo bladder afferent responses to distension in the absence of changes to muscle compliance. Further evaluation revealed that Ca V 3.2 blockers significantly inhibited both low- and high-threshold afferents, decreasing peak responses to distension, and delayed activation thresholds, thereby attenuating bladder afferent responses to both physiological and noxious distension. Nocifensive visceromotor responses to noxious bladder distension in vivo were also significantly reduced by inhibition of Ca V 3 with TTA-A2. Together, these data provide evidence of a major role for Ca V 3.2 in regulating bladder afferent responses to bladder distension and nociceptive signalling to the spinal cord.

An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4.

Activity-based protein profiling (ABPP) is a versatile strategy for identifying and characterizing functional protein sites and compounds for therapeutic development. However, the vast majority of ABPP methods for covalent drug discovery target highly nucleophilic amino acids such as cysteine or lysine. Here, we report a methionine-directed ABPP platform using Redox-Activated Chemical Tagging (ReACT), which leverages a biomimetic oxidative ligation strategy for selective methionine modification. Application of ReACT to oncoprotein cyclin-dependent kinase 4 (CDK4) as a representative high-value drug target identified three new ligandable methionine sites. We then synthesized a methionine-targeting covalent ligand library bearing a diverse array of heterocyclic, heteroatom, and stereochemically rich substituents. ABPP screening of this focused library identified 1oxF11 as a covalent modifier of CDK4 at an allosteric M169 site. This compound inhibited kinase activity in a dose-dependent manner on purified protein and in breast cancer cells. Further investigation of 1oxF11 found prominent cation-π and H-bonding interactions stabilizing the binding of this fragment at the M169 site. Quantitative mass-spectrometry studies validated 1oxF11 ligation of CDK4 in breast cancer cell lysates. Further biochemical analyses revealed cross-talk between M169 oxidation and T172 phosphorylation, where M169 oxidation prevented phosphorylation of the activating T172 site on CDK4 and blocked cell cycle progression. By identifying a new mechanism for allosteric methionine redox regulation on CDK4 and developing a unique modality for its therapeutic intervention, this work showcases a generalizable platform that provides a starting point for engaging in broader chemoproteomics and protein ligand discovery efforts to find and target previously undruggable methionine sites.

The Deadly Toxin Arsenal of the Tree-Dwelling Australian Funnel-Web Spiders.

Australian funnel-web spiders are amongst the most dangerous venomous animals. Their venoms induce potentially deadly symptoms, including hyper- and hypotension, tachycardia, bradycardia and pulmonary oedema. Human envenomation is more frequent with the ground-dwelling species, including the infamous Sydney funnel-web spider (Atrax robustus); although, only two tree-dwelling species induce more severe envenomation. To unravel the mechanisms that lead to this stark difference in clinical outcomes, we investigated the venom transcriptome and proteome of arboreal Hadronyche cerberea and H. formidabilis. Overall, Hadronyche venoms comprised 44 toxin superfamilies, with 12 being exclusive to tree-dwellers. Surprisingly, the major venom components were neprilysins and uncharacterized peptides, in addition to the well-known ω- and δ-hexatoxins and double-knot peptides. The insecticidal effects of Hadronyche venom on sheep blowflies were more potent than Atrax venom, and the venom of both tree- and ground-dwelling species potently modulated human voltage-gated sodium channels, particularly NaV1.2. Only the venom of tree-dwellers exhibited potent modulation of voltage-gated calcium channels. H. formidabilis appeared to be under less diversifying selection pressure compared to the newly adapted tree-dweller, H. cerberea. Thus, this study contributes to unravelling the fascinating molecular and pharmacological basis for the severe envenomation caused by the Australian tree-dwelling funnel-web spiders.

Tips in navigating the diagnostic complexities of chronic inflammatory demyelinating polyradiculoneuropathy.

The 2021 guideline of the European Academy of Neurology/Peripheral Nerve Society on chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) includes important revisions to the previous 2010 guideline. This article highlights the new criteria and recommendations for the differential diagnosis of CIDP. In the revised guideline, the CIDP spectrum has been modified to include typical CIDP and four well-characterized CIDP variants, namely distal, multifocal/focal, motor and sensory CIDP, replacing the term 'atypical' CIDP. To improve the diagnosis of CIDP, the revised guideline attempts to improve the specificity of the diagnostic criteria for typical CIDP and the four CIDP variants. Specific clinical and electrodiagnostic (including both motor and sensory conduction) criteria are provided for typical CIDP and each of the CIDP variants. The levels of diagnostic certainty have been changed to CIDP and possible CIDP, with the removal of probable CIDP (due to the lack of difference in the accuracy of the electrodiagnostic criteria for probable CIDP) and definite CIDP (due to the lack of a gold standard for diagnosis). If the clinical and electrodiagnostic criteria allow only for a diagnosis of possible CIDP, cerebrospinal fluid analysis, nerve ultrasound, nerve magnetic resonance imaging, objective treatment response, and nerve biopsy can be used as supportive criteria to upgrade the diagnosis to CIDP. Although the revised guideline needs to be validated and its strengths and weaknesses assessed, using the guideline will likely improve the accuracy of diagnosis of CIDP and variants of CIDP, and aid in distinguishing CIDP from conditions with similar features.

Transplantation of human neural progenitor cells secreting GDNF into the spinal cord of patients with ALS: a phase 1/2a trial.

Amyotrophic lateral sclerosis (ALS) involves progressive motor neuron loss, leading to paralysis and death typically within 3-5 years of diagnosis. Dysfunctional astrocytes may contribute to disease and glial cell line-derived neurotrophic factor (GDNF) can be protective. Here we show that human neural progenitor cells transduced with GDNF (CNS10-NPC-GDNF) differentiated to astrocytes protected spinal motor neurons and were safe in animal models. CNS10-NPC-GDNF were transplanted unilaterally into the lumbar spinal cord of 18 ALS participants in a phase 1/2a study (NCT02943850). The primary endpoint of safety at 1 year was met, with no negative effect of the transplant on motor function in the treated leg compared with the untreated leg. Tissue analysis of 13 participants who died of disease progression showed graft survival and GDNF production. Benign neuromas near delivery sites were common incidental findings at post-mortem. This study shows that one administration of engineered neural progenitors can provide new support cells and GDNF delivery to the ALS patient spinal cord for up to 42 months post-transplantation.

Diindolylmethane Derivatives: New Selective Blockers for T-Type Calcium Channels.

The natural product indole-3-carbinol (I3C) and its major digestive product 3,3'-diindolylmethane (DIM) have shown clinical promise in multiple forms of cancer including breast cancer. In this study, we explored the calcium channel activity of DIM, its synthetic derivative 3,3'-Diindolylmethanone (DIM-one) and related I3C and DIM-one analogs. For the first time, DIM, DIM-one and analog IX were identified as selective blockers for T-type CaV3.3 (IC50s DIM 2.09 µM; DIM-one 9.07 µM) while compound IX inhibited both CaV3.2 (6.68 µM) and CaV3.3 (IC50 = 3.05 µM) using a FLIPR cell-based assay to measure inhibition of T-type calcium channel window current. Further characterization of DIM by electrophysiology revealed it inhibited inward Ca2+ current through CaV3.1 (IC50 = 8.32 µM) and CaV3.3 (IC50 = 9.63 µM), while IX partially blocked CaV3.2 and CaV3.3 inward Ca2+ current. In contrast, DIM-one preferentially blocked CaV3.1 inward Ca2+ current (IC50 = 1.53 µM). The anti-proliferative activities of these compounds revealed that oxidation of the methylene group of DIM shifted the selectivity of DIMs from breast cancer cell line MCF-7 to colon cancer cell line HT-29.