Amyotrophic lateral sclerosis care and research in the United States during the COVID-19 pandemic: Challenges and opportunities.

Coronavirus disease 2019 has created unprecedented challenges for amyotrophic lateral sclerosis (ALS) clinical care and research in the United States. Traditional evaluations for making an ALS diagnosis, measuring progression, and planning interventions rely on in-person visits that may now be unsafe or impossible. Evidence- and experience-based treatment options, such as multidisciplinary team care, feeding tubes, wheelchairs, home health, and hospice, have become more difficult to obtain and in some places are unavailable. In addition, the pandemic has impacted ALS clinical trials by impairing the ability to obtain measurements for trial eligibility, to monitor safety and efficacy outcomes, and to dispense study drug, as these also often rely on in-person visits. We review opportunities for overcoming some of these challenges through telemedicine and novel measurements. These can reoptimize ALS care and research in the current setting and during future events that may limit travel and face-to-face interactions.

The human gut microbiota in people with amyotrophic lateral sclerosis.

OBJECTIVE: To characterize the gut microbiota in people with amyotrophic lateral sclerosis (ALS) relative to controls and to test the hypothesis that butyrate-producing bacteria are less abundant in the gastrointestinal tracts of people with ALS (PALS). Methods: We conducted a case-control study at Massachusetts General Hospital to compare the gut microbiota in people with ALS to that in controls. Metagenomic shotgun sequencing was performed on DNA extracted from stool samples of 66 people with ALS (PALS), 61 healthy controls (HC), and 12 neurodegenerative controls (NDC). Taxonomic metagenomic profiles were analyzed for shifts in the microbial community structure between the comparator groups using per-feature univariate and multivariate association tests. Results: The relative abundance of the dominant butyrate-producing bacteria Eubacterium rectale and Roseburia intestinalis was significantly lower in ALS patients compared to HC. Adjustment for age, sex, and constipation did not materially change the results. The total abundance of 8 dominant species capable of producing butyrate was also significantly lower in ALS compared to HC (p < 0.001). Conclusions: The levels of several butyrate-producing bacteria, which are important for gut integrity and regulation of inflammation, were lower in people with ALS compared to controls. These findings lend support to the inference that the gut microbiota could be a risk factor for ALS. Further investigations are warranted, preferably earlier in the disease with corresponding dietary collection and a longitudinal design.

Targeting retinoblastoma protein phosphorylation in combination with EGFR inhibition in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains a particularly lethal disease that is resistant to targeted therapies. Tyrosine kinase inhibitors (TKIs), including erlotinib and gefitinib, which block the action of the human epidermal growth factor receptor type 1 receptor, provide small increases in patient survival when administered with gemcitabine. The retinoblastoma (Rb) tumor suppressor protein is an additional target in pancreatic cancer, due to its documented inactivation in PDAC. The present study, using cell number, apoptosis and immunoblotting assays, aimed to evaluate the effects of activation of the Rb tumor suppressor via dephosphorylation by small interfering RNA­mediated phosphatase activation. In the Panc1, MIAPaCa­2 and Capan­2 pancreatic cancer cell lines, and in normal H6c7 cells, the effects of phosphatase activation on Rb were revealed to be dependent on expression of the p16 tumor suppressor, which regulates Rb phosphorylation. Phosphatase activation had no effect on non­transformed pancreatic epithelial cells. When comparing kinase inhibition with phosphatase activation, it was demonstrated that kinase inhibition reduced proliferation, whereas phosphatase activation induced apoptosis. Both treatments together resulted in a greater reduction of pancreatic cancer cells than either treatment alone. In addition, the effects of combination treatment of phosphatase activation with TKIs on cell number and activation of the signal transducer and activator of transcription 3 (STAT3) resistance pathway were determined. The combination of Rb phosphatase activation with TKIs resulted in a greater reduction in cell number compared with either treatment alone, without STAT3 pathway activation. These data suggested that targeting Rb phosphorylation by activating phosphatase may be a rational strategy to inhibit pancreatic tumor cell growth, without activation of acquired resistance.

Dephosphorylation of the Retinoblastoma protein (Rb) inhibits cancer cell EMT via Zeb.

The tumor suppressor Retinoblastoma (Rb) protein is highly phosphorylated in cancer cells largely due to the overexpression of cyclins or the loss of expression of cyclin dependent kinase inhibitors (cdki). Hyperphosphorylation of Rb promotes proliferation, and plays a role in the regulation of apoptosis. Recently, inhibition of cyclin dependent activity toward Rb has been identified as a strategy that has shown clinical efficacy. We utilized a method to induce phosphatase activity toward Rb in cells by shRNA silencing of PNUTS (Phosphatase Nuclear Targeting Subunit) that regulates PP1-mediated dephosphorylation of Rb. In this study, the effect of Rb dephosphorylation on the epithelial to mesenchymal transition (EMT) was determined. The EMT transition is observed in cancer cells that have acquired invasive characteristics. In breast cancer cells grown in 3D Matrigel cultures, MCF7 cells undergo apoptosis in response to Rb dephosphorylation, whereas MDA-MB-231 and Hs578T cells exhibit a reduction in the EMT. Cells devoid of phosphorylated Rb (nontransformed MCF10A and Rb-null MDA-MB-468) lacked any response to PNUTS depletion, showing the effect is Rb-dependent. In addition, these studies showed that Rb dephosphorylation in 3D Matrigel cultures of highly invasive HT1080 cells led to the inhibition of the EMT. Furthermore we observed association between dephosphorylated Rb with ZEB1, a zinc-finger E-box-binding transcription factor that regulates expression of E- and N-cadherins. Finally Rb dephosphorylation led to inhibition of ZEB1 transcriptional activity, this data supports the notion that Rb dephosphorylation modulates the EMT. These studies suggest targeting Rb phosphorylation in mesenchymal cancer cells may decrease invasiveness.