Ribosomal protein eL39 is important for maturation of the nascent polypeptide exit tunnel and proper protein folding during translation.

During translation, nascent polypeptide chains travel from the peptidyl transferase center through the nascent polypeptide exit tunnel (NPET) to emerge from 60S subunits. The NPET includes portions of five of the six 25S/5.8S rRNA domains and ribosomal proteins uL4, uL22, and eL39. Internal loops of uL4 and uL22 form the constriction sites of the NPET and are important for both assembly and function of ribosomes. Here, we investigated the roles of eL39 in tunnel construction, 60S biogenesis, and protein synthesis. We show that eL39 is important for proper protein folding during translation. Consistent with a delay in processing of 27S and 7S pre-rRNAs, eL39 functions in pre-60S assembly during middle nucleolar stages. Our biochemical assays suggest the presence of eL39 in particles at these stages, although it is not visualized in them by cryo-electron microscopy. This indicates that eL39 takes part in assembly even when it is not fully accommodated into the body of pre-60S particles. eL39 is also important for later steps of assembly, rotation of the 5S ribonucleoprotein complex, likely through long range rRNA interactions. Finally, our data strongly suggest the presence of alternative pathways of ribosome assembly, previously observed in the biogenesis of bacterial ribosomal subunits.

A Genome-Wide Functional Screen Identifies Enhancer and Protective Genes for Amyloid Beta-Peptide Toxicity.

Alzheimer's disease (AD) is known to be caused by amyloid ß-peptide (Aß) misfolded into ß-sheets, but this knowledge has not yet led to treatments to prevent AD. To identify novel molecular players in Aß toxicity, we carried out a genome-wide screen in Saccharomyces cerevisiae, using a library of 5154 gene knock-out strains expressing Aß1-42. We identified 81 mammalian orthologue genes that enhance Aß1-42 toxicity, while 157 were protective. Next, we performed interactome and text-mining studies to increase the number of genes and to identify the main cellular functions affected by Aß oligomers (oAß). We found that the most affected cellular functions were calcium regulation, protein translation and mitochondrial activity. We focused on SURF4, a protein that regulates the store-operated calcium channel (SOCE). An in vitro analysis using human neuroblastoma cells showed that SURF4 silencing induced higher intracellular calcium levels, while its overexpression decreased calcium entry. Furthermore, SURF4 silencing produced a significant reduction in cell death when cells were challenged with oAß1-42, whereas SURF4 overexpression induced Aß1-42 cytotoxicity. In summary, we identified new enhancer and protective activities for Aß toxicity and showed that SURF4 contributes to oAß1-42 neurotoxicity by decreasing SOCE activity.

Methyl Jasmonate and Nanoparticles Doped with Methyl Jasmonate affect the Cell Wall Composition of Monastrell Grape Skins.

The structural composition of the cell wall of grape skins is related to the cell wall integrity and subsequent extraction of the different compounds that are contained inside vacuoles and also the cell wall breakdown products. Different reports have established that methyl jasmonate (MeJ) produces changes in the composition of the grape skin cell wall. The use of elicitors to promote the production of secondary metabolites in grapes has been studied in several reports; however, its study linked to nanotechnology is less developed. These facts led us to study the effect of methyl jasmonate (MeJ) and nanoparticles doped with MeJ (nano-MeJ) on the cell walls of Monastrell grapes during three seasons. Both treatments tended to increase cell wall material (CWM) and caused changes in different components of the skin cell walls. In 2019 and 2021, proteins were enlarged in both MeJ and nano-MeJ-treated grapes. A general decrease in total phenolic compounds was detected with both treatments, in addition to an increment in uronic acids when the grapes were well ripened. MeJ and nano-MeJ produced a diminution in the amount of cellulose in contrast to an increase in hemicellulose. It should be noted that the effects with nano-MeJ treatment occurred at a dose 10 times lower than with MeJ treatment.

Different response of proanthocyanidins from Vitis vinifera cv. Monastrell depending on time of elicitor application.

BACKGROUND: Proanthocyanidins (PAs) are phenolic compounds present in skins and seeds of wine grapes and have great implications for plant physiology and wine quality. There are several strategies to increase PA concentration, such as application of elicitors methyl jasmonate (MeJ) and benzothiadiazole (BTH), compounds that can stimulate defence responses like phenolic compound biosynthesis in wine grapes, which have been applied mainly at veraison (beginning of ripening). We recently evaluated the application of MeJ and BTH on Vitis vinifera cv. Monastrell grapes during veraison and mid-ripening (3 weeks after veraison). Grapes treated at mid-ripening showed higher anthocyanin concentrations than those at veraison. In this trial, over two seasons, we evaluated whether time of application (veraison or mid-ripening) of MeJ and BTH on 'Monastrell' grapes is a determining factor in the biosynthesis and composition of PAs in grapes and their subsequent release into wines. RESULTS: Application of elicitors at different ripening times produced significant differences in the PAs of 'Monastrell' grapes, since those treated at mid-ripening recorded a higher PAs concentration in skin and seeds, and then in the wines produced, compared to grapes treated at veraison. CONCLUSION: Results suggest that despite different environmental conditions endured in each of the two seasons evaluated, application of elicitors at mid-ripening of Monastrell grapes could be used to harvest grapes with higher PA concentration, increasing the functional value of the wines, without altering their organoleptic quality. © 2022 Society of Chemical Industry.

Colon adenocarcinoma associated with intestinal sarcoidosis.

Sarcoidosis is a multisystem chronic inflammatory disease of unknown etiology, characterized by non-caseating granulomas. Sarcoidosis has the potential to involve every tissue in the body, which mainly affect the lymphatic system and lungs; gastrointestinal system, and particularly the colon, is an extremely rare location. We report the case of a 64-year-old male with history of pulmonary and cutaneous sarcoidosis diagnosed with neoplasm in the hepatic flexure of the colon and a polyp with high-grade dysplasia in the transverse colon by colonoscopy after a positive fecal occult blood test. The case was presented to a multidisciplinary committee and it was decided to perform a total laparoscopic colectomy and ileorectal anastomosis with histopathological evidence of infiltrating adenocarcinoma and intestinal sarcoidosis with non-caseating granulomas in the appendix, terminal ileum, colon and locoregional lymph nodes. The relationship between colon cancer and sarcoidosis is controversial, with studies showing a possible increased risk of cancer in patients with sarcoidosis, relating it to the chronic proinflammatory state of the disease. In these cases, lymph node involvement is especially important when assessing tumor extension studies, and may lead to changes in staging and, as a consequence, in the therapeutic approach.

The C-terminal tail of ribosomal protein Rps15 is engaged in cytoplasmic pre-40S maturation.

The small ribosomal subunit protein Rps15/uS19 is involved in early nucleolar ribosome biogenesis and subsequent nuclear export of pre-40S particles to the cytoplasm. In addition, the C-terminal tail of Rps15 was suggested to play a role in mature ribosomes, namely during translation elongation. Here, we show that Rps15 not only functions in nucleolar ribosome assembly but also in cytoplasmic pre-40S maturation, which is indicated by a strong genetic interaction between Rps15 and the 40S assembly factor Ltv1. Specifically, mutations either in the globular or C-terminal domain of Rps15 when combined with the non-essential ltv1 null allele are lethal or display a strong growth defect. However, not only rps15 ltv1 double mutants but also single rps15 C-terminal deletion mutants exhibit an accumulation of the 20S pre-rRNA in the cytoplasm, indicative of a cytoplasmic pre-40S maturation defect. Since in pre-40S particles, the C-terminal tail of Rps15 is positioned between assembly factors Rio2 and Tsr1, we further tested whether Tsr1 is genetically linked to Rps15, which indeed could be demonstrated. Thus, the integrity of the Rps15 C-terminal tail plays an important role during late pre-40S maturation, perhaps in a quality control step to ensure that only 40S ribosomal subunits with functional Rps15 C-terminal tail can efficiently enter translation. As mutations in the C-terminal tail of human RPS15 have been observed in connection with chronic lymphocytic leukaemia, it is possible that apart from defects in translation, an impaired late pre-40S maturation step in the cytoplasm could also be a reason for this disease.

Effects of Methyl Jasmonate and Nano-Methyl Jasmonate Treatments on Monastrell Wine Volatile Composition.

The application of methyl jasmonate (MeJ) as an elicitor to enhance secondary metabolites in grapes and wines has been studied, but there is little information about its use in conjunction with nanotechnology and no information about its effects on wine volatile compounds. This led us to study the impact of nanoparticles doped with MeJ (Nano-MeJ, 1mM MeJ) on the volatile composition of Monastrell wines over three seasons, compared with the application of MeJ in a conventional way (10 mM MeJ). The results showed how both treatments enhanced fruity esters in wines regardless of the vintage year, although the increase was more evident when grapes were less ripe. These treatments also achieved these results in 2019 in the cases of 1-propanol, ß-phenyl-ethanol, and methionol, in 2020 in the cases of hexanol and methionol, and in 2021, but only in the case of hexanol. On the other hand, MeJ treatment also increased the terpene fraction, whereas Nano-MeJ, at the applied concentration, did not increase it in any of the seasons. In summary, although not all families of volatile compounds were increased by Nano-MeJ, the Nano-MeJ treatment generally increased the volatile composition to an extent similar to that obtained with MeJ used in a conventional way, but at a 10 times lower dose. Therefore, the use of nanotechnology could be a good option for improving the quality of wines from an aromatic point of view, while reducing the necessary dosage of agrochemicals, in line with more sustainable agricultural practices.

Early-onset severe spinocerebellar ataxia 42 with neurodevelopmental deficits (SCA42ND): Case report, pharmacological trial, and literature review.

Early-onset severe spinocerebellar ataxia 42 with neurodevelopmental deficits (SCA42ND, MIM#604065) is an ultrarare autosomal dominant syndrome related to de novo CACNA1G gain-of-function pathogenic variants. All patients with SCA42ND show cerebellar atrophy and/or hypoplasia on neuroimaging and share common features such as dysmorphic features, global developmental delay, and axial hypotonia, all manifesting within the first year of life. To date, only 10 patients with SCA42ND have been reported with functionally confirmed gain-of-function variants, bearing either of two recurrent pathogenic variants. We describe a girl with congenital ataxia, without epilepsy, and a de novo p.Ala961Thr pathogenic variant in CACNA1G. We review the published subjects with the aim of better characterizing the dysmorphic features that may be crucial for clinical recognition of SCA42ND. Cerebellar atrophy, together with digital anomalies, particularly broad thumbs and/or halluces, should lead to clinical suspicion of this disease. We describe the first pharmacological attempt to treat a patient with SCA42ND using zonisamide, an antiepileptic drug with T-type channel blocker activity, in an off-label indication using an itemized study protocol. No efficacy was observed at the dose tested. However, without pharmacological treatment, she showed a positive evolution in neurodevelopment during the follow-up.

Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses.

Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2.

Atrial Fibrillation in Heart Failure Is Associated with High Levels of Circulating microRNA-199a-5p and 22-5p and a Defective Regulation of Intracellular Calcium and Cell-to-Cell Communication.

MicroRNAs (miRNAs) participate in atrial remodeling and atrial fibrillation (AF) promotion. We determined the circulating miRNA profile in patients with AF and heart failure with reduced ejection fraction (HFrEF), and its potential role in promoting the arrhythmia. In plasma of 98 patients with HFrEF (49 with AF and 49 in sinus rhythm, SR), differential miRNA expression was determined by high-throughput microarray analysis followed by replication of selected candidates. Validated miRNAs were determined in human atrial samples, and potential arrhythmogenic mechanisms studied in HL-1 cells. Circulating miR-199a-5p and miR-22-5p were significantly increased in HFrEF patients with AF versus those with HFrEF in SR. Both miRNAs, but particularly miR-199a-5p, were increased in atrial samples of patients with AF. Overexpression of both miRNAs in HL-1 cells resulted in decreased protein levels of L-type Ca2+ channel, NCX and connexin-40, leading to lower basal intracellular Ca2+ levels, fewer inward currents, a moderate reduction in Ca2+ buffering post-caffeine exposure, and a deficient cell-to-cell communication. In conclusion, circulating miR-199a-5p and miR-22-5p are higher in HFrEF patients with AF, with similar findings in human atrial samples of AF patients. Cells exposed to both miRNAs exhibited altered Ca2+ handling and defective cell-to-cell communication, both findings being potential arrhythmogenic mechanisms.

CACNA1A Mutations Causing Early Onset Ataxia: Profiling Clinical, Dysmorphic and Structural-Functional Findings.

The CACNA1A gene encodes the pore-forming α1A subunit of the voltage-gated CaV2.1 Ca2+ channel, essential in neurotransmission, especially in Purkinje cells. Mutations in CACNA1A result in great clinical heterogeneity with progressive symptoms, paroxysmal events or both. During infancy, clinical and neuroimaging findings may be unspecific, and no dysmorphic features have been reported. We present the clinical, radiological and evolutionary features of three patients with congenital ataxia, one of them carrying a new variant. We report the structural localization of variants and their expected functional consequences. There was an improvement in cerebellar syndrome over time despite a cerebellar atrophy progression, inconsistent response to acetazolamide and positive response to methylphenidate. The patients shared distinctive facial gestalt: oval face, prominent forehead, hypertelorism, downslanting palpebral fissures and narrow nasal bridge. The two α1A affected residues are fully conserved throughout evolution and among the whole human CaV channel family. They contribute to the channel pore and the voltage sensor segment. According to structural data analysis and available functional characterization, they are expected to exert gain- (F1394L) and loss-of-function (R1664Q/R1669Q) effect, respectively. Among the CACNA1A-related phenotypes, our results suggest that non-progressive congenital ataxia is associated with developmental delay and dysmorphic features, constituting a recognizable syndromic neurodevelopmental disorder.

Application of Elicitors in Two Ripening Periods of Vitis vinifera L. cv Monastrell: Influence on Anthocyanin Concentration of Grapes and Wines.

In recent years, it has been demonstrated that the application of elicitors such as methyl-jasmonate (MeJ) and benzothiadiazole (BTH) to wine grapes can increase their phenolic and aromatic compounds if they are treated at the beginning of ripening (veraison). However, the veraison period is short, and it is not always possible to apply the treatments in a few days. Therefore, it would be of great interest to optimize the moment of elicitor application or extend the treatment period. The aim of this paper was to analyze during two consecutive years (2016-2017) the foliar application of MeJ, BTH, and a combination of both, during two different ripening periods of Monastrell grapes (veraison and mid-ripening), and determine the more appropriate moment to increase the concentration of anthocyanins. To carry out this aim, analysis of anthocyanins by HPLC in grapes and wines was mainly performed. The most suitable period for the application of MeJ, BTH, and MeJ + BTH was at mid-ripening, since the grapes showed a greater accumulation of anthocyanins at harvest. However, the MeJ + BTH treatment applied during veraison also obtained similar results, which would allow extending the application period if necessary. However, the increase in the anthocyanin content of grapes was not reflected in all the wines, which may have been due to reinforcement of the skin cell wall as a result of the application of elicitors. Further analysis is needed to improve the maceration process of the Monastrell grapes and the extraction of the anthocyanins that were increased by the treatments applied in the vineyard.

Applicability of 3D-printed models in hepatobiliary surgey: results from "LIV3DPRINT" multicenter study.

BACKGROUND: Hepatobiliary resections are challenging due to the complex liver anatomy. Three-dimensional printing (3DP) has gained popularity due to its ability to produce anatomical models based on the characteristics of each patient. METHODS: A multicenter study was conducted on complex hepatobiliary tumours. The endpoint was to validate 3DP model accuracy from original image sources for application in the teaching, patient-communication, and planning of hepatobiliary surgery. RESULTS: Thirty-five patients from eight centers were included. Process testing between 3DP and CT/MRI presented a considerable degree of similarity in vascular calibers (0.22 ± 1.8 mm), and distances between the tumour and vessel (0.31 ± 0.24 mm). The Dice Similarity Coefficient was 0.92, with a variation of 2%. Bland-Altman plots also demonstrated an agreement between 3DP and the surgical specimen with the distance of the resection margin (1.15 ± 1.52 mm). Professionals considered 3DP at a positive rate of 0.89 (95%CI; 0.73-0.95). According to student's distribution a higher success rate was reached with 3DP (median:0.9, IQR: 0.8-1) compared with CT/MRI or 3D digital imaging (P = 0.01). CONCLUSION: 3DP hepatic models present a good correlation compared with CT/MRI and surgical pathology and they are useful for education, understanding, and surgical planning, but does not necessarily affect the surgical outcome.

Rare CACNA1A mutations leading to congenital ataxia.

Human mutations in the CACNA1A gene that encodes the pore-forming α1A subunit of the voltage-gated CaV2.1 (P/Q-type) Ca2+ channel cause multiple neurological disorders including sporadic and familial hemiplegic migraine, as well as cerebellar pathologies such as episodic ataxia, progressive ataxia, and early-onset cerebellar syndrome consistent with the definition of congenital ataxia (CA), with presentation before the age of 2 years. Such a pathological role is in accordance with the physiological relevance of CaV2.1 in neuronal tissue, especially in the cerebellum. This review deals with the report of the main clinical features defining CA, along with the presentation of an increasing number of CACNA1A genetic variants linked to this severe cerebellar disorder in the context of Ca2+ homeostasis alteration. Moreover, the review describes each pathological mutation according to structural location and known molecular and cellular functional effects in both heterologous expression systems and animal models. In view of this information in correlation with the clinical phenotype, we take into consideration different pathomechanisms underlying the observed motor dysfunction in CA patients carrying CACNA1A mutations. Present therapeutic management in CA and options for the development of future personalized treatment based on CaV2.1 dysfunction are also discussed.

Role of the 40S beak ribosomal protein eS12 in ribosome biogenesis and function in Saccharomyces cerevisiae.

In eukaryotes, the beak structure of 40S subunits is formed by the protrusion of the 18S rRNA helix 33 and three ribosomal proteins: eS10, eS12 and eS31. The exact role of these proteins in ribosome biogenesis is not well understood. While eS10 is an essential protein encoded by two paralogous genes in Saccharomyces cerevisiae, eS12 and eS31 are not essential proteins encoded by the single-copy genes RPS12 and UBI3, respectively. Here, we have analysed the contribution of yeast eS12 to ribosome biogenesis and compared it with that of eS31. Polysome analysis reveals that deletion of either RPS12 or UBI3 results in equivalent 40S deficits. Analysis of pre-rRNA processing indicates that eS12, akin to eS31, is required for efficient processing of 20S pre-rRNA to mature 18S rRNA. Moreover, we show that the 20S pre-rRNA accumulates within cytoplasmic pre-40S particles, as deduced from FISH experiments and the lack of nuclear retention of 40S subunit reporter proteins, in rps12∆ and ubi3∆ cells. However, these particles containing 20S pre-rRNA are not efficiently incorporated into polyribosomes. We also provide evidence for a genetic interaction between eS12 or eS31 and the late-acting 40S assembly factors Enp1 and Ltv1, which appears not to be linked to the dynamics of their association with or release from pre-40S particles in the absence of either eS12 or eS31. Finally, we show that eS12- and eS31-deficient ribosomes exhibit increased levels of translational misreading. Altogether, our data highlight distinct important roles of the beak region during ribosome assembly and function.

Ribosomal protein L14 contributes to the early assembly of 60S ribosomal subunits in Saccharomyces cerevisiae.

The contribution of most ribosomal proteins to ribosome synthesis has been quite well analysed in Saccharomyces cerevisiae. However, few yeast ribosomal proteins still await characterization. Herein, we show that L14, an essential 60S ribosomal protein, assembles in the nucleolus at an early stage into pre-60S particles. Depletion of L14 results in a deficit in 60S subunits and defective processing of 27SA2 and 27SA3 to 27SB pre-rRNAs. As a result, 27S pre-rRNAs are subjected to turnover and export of pre-60S particles is blocked. These phenotypes likely appear as the direct consequence of the reduced pre-60S particle association not only of L14 upon its depletion but also of a set of neighboring ribosomal proteins located at the solvent interface of 60S subunits and the adjacent region surrounding the polypeptide exit tunnel. These pre-60S intermediates also lack some essential trans-acting factors required for 27SB pre-rRNA processing but accumulate practically all factors required for processing of 27SA3 pre-rRNA. We have also analysed the functional interaction between the eukaryote-specific carboxy-terminal extensions of the neighboring L14 and L16 proteins. Our results indicate that removal of the most distal parts of these extensions cause slight translation alterations in mature 60S subunits.

Aromatic Characterization of New White Wine Varieties Made from Monastrell Grapes Grown in South-Eastern Spain.

The aromatic profile of a wine is one of the main characteristics appreciated by consumers. Due to climate change, vineyards need to adapt to new conditions, and one of the strategies that might be followed is to develop new white varieties from Monastrell and other cultivars by means of intervarietal crosses, since white varieties are a minority in south-eastern Spain. Such crosses have already been obtained and have been seen to provide quality white wines of high acidity and with a good aromatic composition. To confirm this, a quantitative analysis was carried out during two vintages (2018 and 2019) in order to study and compare the volatile composition of Verdejo (V) wine with the aromatic composition of several wines made from different crosses between Cabernet Sauvignon (C), Syrah (S), Tempranillo (T), and Verdejo (V) with Monastrell (M), by means of headspace SPME-GC-MS analysis. Wine volatile compounds (alcohols, volatile acids, ethyl esters, terpenes, norisoprenoids, and two other compounds belonging to a miscellaneous group) were identified and quantified using a HS-SPME-GS-MS methodology. An additional sensory analysis was carried out by a qualified tasting panel in order to characterize the different wines. The results highlighted how the crosses MT103, MC69, and MC180 showed significant differences from and better quality than the Verdejo wine. These crosses produced higher concentrations of several aromatic families analyzed, which was supported by the views of the tasting panel, thus confirming their excellent aromatic potential as cultivars for producing grapes well adapted to this area for making white wines.

Study of aromatic profile of different crosses of Monastrell white wines.

BACKGROUND: Aromatic compounds are responsible for the final quality of wines. A semi-quantitative analysis was carried out during two consecutive seasons aiming to determine the volatile composition of 12 new white crosses obtained between Monastrell (M) and other varieties, such as Cabernet Sauvignon (C), Syrah (S) and Tempranillo (T) (MC10, MC180, MC9, MC69, MS30, MS33, MS82, MT103, MT19, MV11, MV67 and MV7), using a methodology based on gas chromatography-solid phase microextraction-mass spectrometry. RESULTS: On the one hand, 30 aromatic compounds were identified belonging to different chemical groups (alcohols, acids, terpenes, norisoprenoids and esters). The results showed how some crosses presented significant differences with respect to their parental. For example, in 2016, Monastrell and Cabernet Sauvignon showed high concentration of alcohols, acids and some terpenes, whereas the corresponding crosses showed a predominance of aromas belonging to esters. In 2017, as a result of edaphoclimatic conditions, the white crosses had higher concentrations of esters and acids. In addition, Monastrell and Cabernet Sauvignon showed similar concentrations of alcohols compared to 2016. On the other hand, sensorial analyses confirmed these results, so that mint and peppermint aromas and a fresh quality were detected in MC69 wine, especially in 2016, and fruity and acid aromas were detected in MC180 wine, which gave it a wide ranging complexity and aromatic potential. CONCLUSION: The present study reports the first investigation of the volatile composition and sensory characteristics of directed crosses white wines obtained from Monastrell and other varieties, such as Cabernet Sauvignon, Syrah, Tempranillo and Verdejo. The results obtained indicate that the use of some of these white crosses could be a good option for growing them in this Mediterranean area as a result of the contribution of a good quality in the wine aroma. © 2019 Society of Chemical Industry.

Treatment of a chronic anal fissure with a botulin toxin A injection and fissurectomy.

INTRODUCTION: pharmacological treatment of a chronic anal fissure (CAF) achieves healing in half of cases and lateral internal sphincterotomy (LIS) is the definite treatment. The objective of this study was to assess the combination of fissurectomy and botulin toxin A (BTA) injection. METHODS: this was a retrospective study of 54 patients with anal sphincter hypertonia and CAF treated with an injection of BAT and fissurectomy, after an unsuccessful management with topical nitroglycerin (NGT) for eight weeks. Fissurectomy and an injection of BTA (33 or 50 units) in the internal anal sphincter was performed during the same session. The main outcome measure was the healing rate, with incontinence and the need of LIS as secondary outcomes. RESULTS: two patients were excluded from the study, one due to Crohn's disease and the other was lost to follow-up. Of the 52 patients included in the study, there were 36 females (70%) and 16 (30%) males, with a mean age of 49 years (range 22-75). Fissure healing was initially achieved in 49 patients (94.2%) and LIS was required in the remaining three patients (5.8%). After initial healing, 18 patients (34.7%) developed 23 recurrences at a mean time of 27 months (5-83 months). Of these patients, healing with conservative sphincter measures was obtained in eleven cases (NGT in eight and repeat fissurectomy and BAT in three); two patients are currently under treatment with NGT and five underwent LIS. CONCLUSIONS: BTA injection associated with fissurectomy is a safe and effective procedure in patients with CAF, avoiding the need of LIS in a high percentage of patients.

Stroke-Like Episodes and Cerebellar Syndrome in Phosphomannomutase Deficiency (PMM2-CDG): Evidence for Hypoglycosylation-Driven Channelopathy.

Stroke-like episodes (SLE) occur in phosphomannomutase deficiency (PMM2-CDG), and may complicate the course of channelopathies related to Familial Hemiplegic Migraine (FHM) caused by mutations in CACNA1A (encoding CaV2.1 channel). The underlying pathomechanisms are unknown. We analyze clinical variables to detect risk factors for SLE in a series of 43 PMM2-CDG patients. We explore the hypothesis of abnormal CaV2.1 function due to aberrant N-glycosylation as a potential novel pathomechanism of SLE and ataxia in PMM2-CDG by using whole-cell patch-clamp, N-glycosylation blockade and mutagenesis. Nine SLE were identified. Neuroimages showed no signs of stroke. Comparison of characteristics between SLE positive versus negative patients' group showed no differences. Acute and chronic phenotypes of patients with PMM2-CDG or CACNA1A channelopathies show similarities. Hypoglycosylation of both CaV2.1 subunits (α1A and α2α) induced gain-of-function effects on channel gating that mirrored those reported for pathogenic CACNA1A mutations linked to FHM and ataxia. Unoccupied N-glycosylation site N283 at α1A contributes to a gain-of-function by lessening CaV2.1 inactivation. Hypoglycosylation of the α2δ subunit also participates in the gain-of-function effect by promoting voltage-dependent opening of the CaV2.1 channel. CaV2.1 hypoglycosylation may cause ataxia and SLEs in PMM2-CDG patients. Aberrant CaV2.1 N-glycosylation as a novel pathomechanism in PMM2-CDG opens new therapeutic possibilities.