Heterogeneous A40926 self-resistance profile in nonomuraea gerenzanensis population informs strain improvement

5noopenNonomuraea gerenzanensis ATCC 39727 produces the glycopeptide antibiotic A40926, which is the natural precursor of the semi-synthetic, last-resort drug dalbavancin. To reduce the cost of dalbavancin production, it is mandatory to improve the productivity of the producing strain. Here, we report that the exposure of N. gerenzanensis wild-type population to sub-inhibitory concentrations of A40926 led to the isolation of differently resistant phenotypes to which a diverse A40926 productivity was associated. The most resistant population (G, grand colonies) represented at least the 20% of the colonies growing on 2 µg/mL of A40926. It showed a stable phenotype after sub-culturing and a homogeneous profile of self-resistance to A40926 in population analysis profile (PAP) experiments. The less resistant population (P, petit) was represented by slow-growing colonies to which a lower A40926 productivity was associated. At bioreactor scale, the G variant produced twice more than the wild-type (ca. 400 mg/L A40926 versus less than 200 mg/L, respectively), paving the way for a rational strain improvement based on the selection of increasingly self-resistant colonies.openElisa Binda, Francesca Berini, Flavia Marinelli, Adriana Bava, Fabrizio BeltramettiBinda, Elisa; Berini, Francesca; Marinelli, Flavia; Bava, Adriana; Beltrametti, Fabrizi

Increased responsiveness at the cerebellar input stage in the PRRT2 knockout model of paroxysmal kinesigenic dyskinesia

PRoline-Rich Transmembrane protein-2 (PRRT2) is a recently described neuron-specific type-2 integral membrane protein with a large cytosolic N-terminal domain that distributes in presynaptic and axonal domains where it interacts with several presynaptic proteins and voltage-gated Na+ channels. Several PRRT2 mutations are the main cause of a wide and heterogeneous spectrum of paroxysmal disorders with a loss-of-function pathomechanism. The highest expression levels of PRRT2 in brain occurs in cerebellar granule cells (GCs) and cerebellar dysfunctions participate in the dyskinetic phenotype of PRRT2 knockout (KO) mice. We have investigated the effects of PRRT2 deficiency on the intrinsic excitability of GCs and the input-output relationships at the mossy fiber-GC synapses. We show that PRRT2 KO primary GCs display increased expression of Na+ channels, increased amplitude of Na+ currents and increased length of the axon initial segment, leading to an overall enhancement of intrinsic excitability. In acute PRRT2 KO cerebellar slices, GCs were more prone to action potential discharge in response to mossy fiber activation and exhibited an enhancement of transient and persistent Na+ currents, in the absence of changes at the mossy fiber-GC synapses. The results support a key role of PRRT2 expressed in GCs in the physiological regulation of the excitatory input to the cerebellum and are consistent with a major role of a cerebellar dysfunction in the pathogenesis of the PRRT2-linked paroxysmal pathologies

Guidelines for the Treatment of Abdominal Abscesses in Acute Diverticulitis: An Umbrella Review

Background: This systematic umbrella review aims to investigate and provide an analysis of guidelines regarding the treatment of diverticular abscesses. Material and methods: A systematic literature search was performed using the Cochrane Overviews of Reviews model and the 'Clinical Practice Guidelines'; at the end of initial search, only 12 guidelines were included in this analysis. The quality of the guidelines was assessed by adopting the "Appraisal of Guidelines for Research and Evaluation II" (AGREE II). The comparative analysis of these guidelines has highlighted the presence of some differences regarding the recommendations on the treatment of diverticular abscesses. In particular, there are some controversies about the diameter of abscess to be used in order to decide between medical treatment and percutaneous drainage. Different guidelines propose different abscess diameter cutoffs, such as 3 cm, 4-5 cm, or 4 cm, for distinguishing between small and large abscesses. Conclusions: Currently, different scientific societies recommend that diverticular abscesses with diameters larger than 3 cm should be considered for percutaneous drainage whereas abscesses with diameters smaller than 3 cm could be appropriately treated by medical therapy with antibiotics; only a few guidelines suggest the use of percutaneous drainage for abscesses with a diameter greater than 4 cm. The differences among guidelines are the consequence of the different selection of scientific evidence. In conclusion, our evaluation has revealed the importance of seeking new scientific evidence with higher quality to either confirm, reinforce or potentially weaken the existing recommendations from different societies

An activator of voltage-gated K+ channels Kv1.1 as a therapeutic candidate for episodic ataxia type 1

Loss-of-function mutations in the KCNA1(Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by KCNA1 mutations has been clinically approved. Here, we uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. We show that through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a knock-in mouse model of EA1 and restored the neuromuscular transmission and climbing ability in Shaker (Kv1.1) mutant Drosophila melanogaster flies (Sh5). By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery

Human neural stem cell transplantation in ALS: initial results from a phase I trial

We report the initial results from a phase I clinical trial for ALS. We transplanted GMP-grade, fetal human neural stem cells from natural in utero death (hNSCs) into the anterior horns of the spinal cord to test for the safety of both cells and neurosurgical procedures in these patients. The trial was approved by the Istituto Superiore di Sanit\ue0 and the competent Ethics Committees and was monitored by an external Safety Board

Inhibition promotes long-term potentiation at cerebellar excitatory synapses.

The ability of the cerebellar cortex to learn from experience ensures the accuracy of movements and reflex adaptation, processes which require long-term plasticity at granule cell (GC) to Purkinje neuron (PN) excitatory synapses. PNs also receive GABAergic inhibitory inputs via GCs activation of interneurons; despite the involvement of inhibition in motor learning, its role in long-term plasticity is poorly characterized. Here we reveal a functional coupling between ionotropic GABAA receptors and low threshold CaV3 calcium channels in PNs that sustains calcium influx and promotes long-term potentiation (LTP) at GC to PN synapses. High frequency stimulation induces LTP at GC to PN synapses and CaV3-mediated calcium influx provided that inhibition is intact; LTP is mGluR1, intracellular calcium store and CaV3 dependent. LTP is impaired in CaV3.1 knockout mice but it is nevertheless recovered by strengthening inhibitory transmission onto PNs; promoting a stronger hyperpolarization via GABAA receptor activation leads to an enhanced availability of an alternative Purkinje-expressed CaV3 isoform compensating for the lack of CaV3.1 and restoring LTP. Accordingly, a stronger hyperpolarization also restores CaV3-mediated calcium influx in PNs from CaV3.1 knockout mice. We conclude that by favoring CaV3 channels availability inhibition promotes LTP at cerebellar excitatory synapses.journal article2016 Sep 192016 09 19importe

Gabapentin treatment in a patient with KCNQ2 developmental epileptic encephalopathy

De novo variants in KCNQ2 encoding for Kv7.2 voltage-dependent neuronal potassium (K+) channel subunits are associated with developmental epileptic encephalopathy (DEE). We herein describe a the clinical and electroencephalographic (EEG) features of a child with early-onset DEE caused by the novel KCNQ2 p.G310S variant. In vitro experiments demonstrated that the mutation induces loss-of-function effects on the currents produced by channels incorporating mutant subunits; these effects were counteracted by the selective Kv7 opener retigabine and by gabapentin, a recently described Kv7 activator. Given these data, the patient started treatment with gabapentin, showing a rapid and sustained clinical and EEG improvement over the following months. Overall, these results suggest that gabapentin can be regarded as a precision therapy for DEEs due to KCNQ2 loss-of-function mutations

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5–11 December, to 17.5% (25/143 samples) in the week 12–18, to 65.9% (89/135 samples) in the week 19–25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased fromone in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool