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BackgroundThe Covid-19 pandemic has emerged as the leading public health challenge of our time (20th century). While vaccinations have finally blunted the death rate, concern has remained about more virulent forms highlighting the need for alternative approaches. Epidemiological studies indicate that physical activity has been shown to decrease the risk of infection of some respiratory viruses. Part of the salutary effects of exercise is believed to be through the elaboration of cytokines by contracting skeletal muscles (termed myokines). The objective of this study was to investigate whether exercise-induced myokines would mitigate the SARS-CoV-2 infectivity of the bronchial epithelium through modulating the SARS-CoV-2 Covid-19 receptor (angiotensin-converting enzyme 2 -ACE2) its priming enzyme, transmembrane serine protease 2 (TMPRSS2).MethodsWe utilized a cell culture model of exercise to generate myokines by differentiating C2C12 cells into myotubules and inducing them to contract via low-frequency electric pulse stimulation. Condition media was concentrated via centrifugation and applied to human immortalized human bronchial epithelium cell line (6HBE14o) along with conditioned media from unstimulated myotubules as controls. Following exposure to myokines, the 16HBE14o cells were harvested and subjected to quantitative RT-PCR and Enzyme-Linked Immunosorbent Assay (ELISA) for assessment of mRNA and protein levels of ACE2 and TMPRSS2, respectively. Pilot proteomic data was performed with isotope barcoding and mass spectroscopy.ResultsQuantitative Real-Time PCR of 16HBE14o with 48 h treated unstimulated vs. stimulated myokine treatment revealed a reduction of ACE2 and TMPRSS2 mRNA by 32% (pConclusionsThe current study explores a novel approach of a modified exercise cell culture system and uses ACE2 and TMPRSS2 as a surrogate marker of SARS-CoV-2 infectivity. In conclusion, we demonstrated biological data supporting exercise's protective effect against Covid-19. These further strengthen myokines' beneficial role as potential therapeutic targets against SARS-CoV-2 and similar viruses albeit these preliminary cell culture studies will require future validation in animal models.

[This corrects the article DOI: 10.1371/journal.pone.0197427.].

PURPOSE:Hepatocellular carcinoma (HCC) results from chronic inflammation/cirrhosis. Unfortunately, despite use of radiological/serological screening techniques, HCC ranks as a leading cause of cancer deaths. Our group has used alterations in high order chromatin as a marker for field carcinogenesis and hence risk for a variety of cancers (including colon, lung, prostate, ovarian, esophageal). In this study we wanted to address whether these chromatin alterations occur in HCC and if it could be used for risk stratification. EXPERIMENTAL DESIGN:A case control study was performed in patients with cirrhosis who went on to develop HCC and patients with cirrhosis who did not develop cancer. We performed partial wave spectroscopic microscopy (PWS) which measures nanoscale alterations on formalin fixed deparaffinized liver biopsy specimens, 17 progressors and 26 non-progressors. Follow up was 2089 and 2892 days, respectively. RESULTS:PWS parameter disorder strength Ld were notably higher for the progressors (Ld = 1.47 ± 0.76) than the non-progressors (Ld = 1.00 ± 0.27) (p = 0.024). Overall, the Cohen's d effect size was 0.907 (90.7%). AUROC analysis yielded an area of 0.70. There was no evidence of confounding by gender, age, BMI, smoking status and race. CONCLUSIONS:High order chromatin alterations, as detected by PWS, is altered in pre-malignant hepatocytes with cirrhosis and may predict future risk of HCC.

Chemoprevention represents an attractive modality against colorectal cancer (CRC) although widespread clinical implementation of promising agents (e.g. aspirin/NSAIDS) have been stymied by both suboptimal efficacy and concerns over toxicity. This highlights the need for better agents. Several groups, including our own, have reported that the over-the-counter laxative polyethylene glycol (PEG) has remarkable efficacy in rodent models of colon carcinogenesis. In this study, we undertook the first randomized human trial to address the role of PEG in prevention of human colonic neoplasia. This was a double-blind, placebo-controlled, three-arm trial where eligible subjects were randomized to 8g PEG-3350 (n = 27) or 17g PEG-3350 (n = 24), or placebo (n = 24; maltodextrin) orally for a duration of six months. Our initial primary endpoint was rectal aberrant crypt foci (ACF) but this was changed during protocol period to rectal mucosal epidermal growth factor receptor (EGFR). Of the 87 patients randomized, 48 completed study primary endpoints and rectal EGFR unchanged PEG treatment. Rectal ACF had a trend suggesting potentially reduction with PEG treatment (pre-post change 1.7 in placebo versus -0.3 in PEG 8+ 17g doses, p = 0.108). Other endpoints (proliferation, apoptosis, expression of SNAIL and E-cadherin), previously noted to be modulated in rodent models, appeared unchanged with PEG treatment in this clinical trial. We conclude that PEG was generally well tolerated with the trial failing to meet primary efficacy endpoints. However, rectal ACFs demonstrated a trend (albeit statistically insignificant) for suppression with PEG. Moreover, all molecular assays including EGFR were unaltered with PEG underscoring issues with lack of translatability of biomarkers from preclinical to clinical trials. This data may provide the impetus for future clinical trials on PEG using more robust biomarkers of chemoprevention. TRIAL REGISTRATION:ClinicalTrials.gov NCT00828984.

To determine whether nano-architectural interrogation of prostate field carcinogenesis can be used to predict prognosis in patients with early stage (Gleason 6) prostate cancer (PCa), which is mostly indolent but frequently unnecessarily treated.We previously developed partial wave spectroscopic microscopy (PWS) that enables quantification of the nanoscale intracellular architecture (20-200 nm length scale) with remarkable accuracy. We adapted this technique to assess prostate needle core biopsies in a case control study from men with Gleason 6 disease who either progressed (n = 20) or remained indolent (n = 18) over a ~3 year follow up. We measured the parameter disorder strength (Ld) characterizing the spatial heterogeneity of the nanoscale cellular structure and nuclear morphology from the microscopically normal mucosa ~150 histologically normal epithelial cells.There was a profound increase in nano-architectural disorder between progressors and non-progressors. Indeed, the Ld from future progressors was dramatically increased when compared to future non-progressors (1±0.065 versus 1.30±0.0614, respectively p = 0.002). The area under the receiver operator characteristic curve (AUC) was 0.79, yielding a sensitivity of 88% and specificity of 72% for discriminating between progressors and non-progressors. This was not confounded by demographic factors (age, smoking status, race, obesity), thus supporting the robustness of the approach.We demonstrate, for the first time, that nano-architectural alterations occur in prostate cancer field carcinogenesis and can be exploited to predict prognosis of early stage PCa. This approach has promise in addressing the clinically vexing dilemma of management of Gleason 6 PCa and may provide a paradigm for dealing with the larger issue of cancer overdiagnosis.

Lung cancer remains the leading cause of cancer deaths in the US with >150,000 deaths per year. In order to more effectively reduce lung cancer mortality, more sophisticated screening paradigms are needed. Previously, our group demonstrated the use of low-coherence enhanced backscattering (LEBS) spectroscopy to detect and quantify the micro/nano-architectural correlates of colorectal and pancreatic field carcinogenesis. In the lung, the buccal (cheek) mucosa has been suggested as an excellent surrogate site in the "field of injury". We, therefore, wanted to assess whether LEBS could similarly sense the presence of lung. To this end, we applied a fiber-optic LEBS probe to a dataset of 27 smokers without diagnosed lung cancer (controls) and 46 with lung cancer (cases), which was divided into a training and a blinded validation set (32 and 41 subjects, respectively). LEBS readings of the buccal mucosa were taken from the oral cavity applying gentle contact. The diagnostic LEBS marker was notably altered in patients harboring lung cancer compared to smoking controls. The prediction rule developed on training set data provided excellent diagnostics with 94% sensitivity, 80% specificity, and 95% accuracy. Applying the same threshold to the blinded validation set yielded 79% sensitivity and 83% specificity. These results were not confounded by patient demographics or impacted by cancer type or location. Moreover, the prediction rule was robust across all stages of cancer including stage I. We envision the use of LEBS as the first part of a two-step paradigm shift in lung cancer screening in which patients with high LEBS risk markers are funnelled into more invasive screening for confirmation.

We previously reported the utility of Low-Coherence Enhanced Backscattering (LEBS) Spectroscopy in detecting optical changes in uninvolved rectal mucosa, changes that are indicative of the presence of advanced colorectal adenomas elsewhere in the colon (field carcinogenesis). We hypothesized that the alterations in optical signatures are due to structural changes in colonocytes. To elucidate those colonocyte changes, we used LEBS and an early time point in an animal model of colorectal field carcinogenesis--rats treated with azoxymethane (AOM). Changes in LEBS markers in intact mucosa from AOM-treated rats could be at least partially attributed to changes in colonocytes. To investigate the molecular mechanisms underlying the colonocyte abnormalities in premalignant colon, we took a candidate approach. We compared expression profiles of genes implicated directly or indirectly in cytoskeletal dysregulation in colorectal tissues from saline-treated versus AOM-treated rats. Our data suggest that a number of genes known to affect colon tumorigenesis are up-regulated in colonocytes, and genes previously reported to be tumor suppressors in metastatic cancer are down-regulated in colonocytes, despite the colonocytes being histologically normal. To further understand the role of the cytoskeleton in generating changes in optical markers of cells, we used pharmacological disruption (using colchicine) of the cytoskeleton. We found that differences in optical markers (between AOM- and control-treated rats) were negated by the disruption, suggesting cytoskeletal involvement in the optical changes. These studies provide significant insights into the micro-architectural alterations in early colon carcinogenesis, and may enable optimization of both bio-photonic and molecular risk stratification techniques to personalize colorectal cancer screening.

Normal cell function is dependent on the proper maintenance of chromatin structure. Regulation of chromatin structure is controlled by histone modifications that directly influence chromatin architecture and genome function. Specifically, the histone deacetylase (HDAC) family of proteins modulate chromatin compaction and are commonly dysregulated in many tumors, including colorectal cancer (CRC). However, the role of HDAC proteins in early colorectal carcinogenesis has not been previously reported. We found HDAC1, HDAC2, HDAC3, HDAC5, and HDAC7 all to be up-regulated in the field of human CRC. Furthermore, we observed that HDAC2 up-regulation is one of the earliest events in CRC carcinogenesis and observed this in human field carcinogenesis, the azoxymethane-treated rat model, and in more aggressive colon cancer cell lines. The universality of HDAC2 up-regulation suggests that HDAC2 up-regulation is a novel and important early event in CRC, which may serve as a biomarker. HDAC inhibitors (HDACIs) interfere with tumorigenic HDAC activity; however, the precise mechanisms involved in this process remain to be elucidated. We confirmed that HDAC inhibition by valproic acid (VPA) targeted the more aggressive cell line. Using nuclease digestion assays and transmission electron microscopy imaging, we observed that VPA treatment induced greater changes in chromatin structure in the more aggressive cell line. Furthermore, we used the novel imaging technique partial wave spectroscopy (PWS) to quantify nanoscale alterations in chromatin. We noted that the PWS results are consistent with the biological assays, indicating a greater effect of VPA treatment in the more aggressive cell type. Together, these results demonstrate the importance of HDAC activity in early carcinogenic events and the unique role of higher-order chromatin structure in determining cell tumorigenicity.

Head and neck squamous cell carcinoma (HNSCC) is a major cause of morbidity and mortality underscoring the need for safe and effective chemopreventive strategies. Targeting epidermal growth factor receptor (EGFR) is attractive in that it is an early critical event in HNSCC pathogenesis. However, current agents lack efficacy or have unacceptable toxicity. Several groups have demonstrated that the over-the-counter medication, polyethylene glycol (PEG) has remarkable chemopreventive efficacy against colon carcinogenesis. Importantly, we reported that this effect is mediated through EGFR internalization/degradation. In the current study, we investigated the chemopreventive efficacy of this agent against HNSCC, using both the well validated animal model 4-NQO (4-nitroquinoline 1-oxide) rat model and cell culture with the human HNSCC cell line SCC-25. We demonstrated that daily topical application of 10% PEG-8000 in the oral cavity (tongue and cavity wall) post 4NQO initiation resulted in a significant reduction in tumor burden (both, tumor size and tumors/tumor bearing rat) without any evidence of toxicity. Immunohistochemical studies depicted decreased proliferation (number of Ki67-positive cells) and reduced expression of EGFR and its downstream effectors cyclin D1 in the tongue mucosa of 4NQO-rats treated with PEG. We showed that EGFR was also markedly downregulated in SCC-25 cells by PEG-8000 with a concomitant induction of G1-S phase cell-cycle arrest, which was potentially mediated through upregulated p21(cip1/waf1). In conclusion, we demonstrate, for the first time, that PEG has promising efficacy and safety as a chemopreventive efficacy against oral carcinogenesis.

Colorectal cancer (CRC) screening tests often have a trade-off between efficacy and patient acceptability/cost. Fecal tests (occult blood, methylation) engender excellent patient compliance but lack requisite performance underscoring the need for better population screening tests. We assessed the utility of microRNAs (miRNAs) as markers of field carcinogenesis and their potential role for CRC screening using the azoxymethane (AOM)-treated rat model. We found that 63 miRNAs were upregulated and miR-122, miR-296-5p and miR-503# were downregulated in the uninvolved colonic mucosa of AOM rats. We monitored the expression of selected miRNAs in colonic biopsies of AOM rats at 16 weeks and correlated it with tumor development. We noted that the tumor bearing rats had significantly greater miRNA modulation compared to those without tumors. The miRNAs showed good diagnostic performance with an area under the receiver operator curve (AUROC) of >0.7. We also noted that the miRNA induction in the colonic mucosa was mirrorred in the mucus layer fecal colonocytes isolated from AOM rat stool and the degree of miRNA induction was greater in the tumor bearing rats compared to those without tumors. Lastly, we also noted significant miRNA modulation in the Pirc rats- the genetic model of colon carcinogenesis, both in the uninvolved colonic mucosa and the fecal colonocytes. We thus demonstrate that miRNAs are excellent markers of field carcinogenesis and could accurately predict future neoplasia. Based on our results, we propose an accurate, inexpensive, non-invasive miRNA test for CRC risk stratification based on rectal brushings or from abraded fecal colonocytes.