Deciphering Bacterial and Archaeal Transcriptional Dark Matter and Its Architectural Complexity.

Transcripts are potential therapeutic targets, yet bacterial transcripts remain biological dark matter with uncharacterized biodiversity. We developed and applied an algorithm to predict transcripts for Escherichia coli K12 and E2348/69 strains (Bacteria:gamma-Proteobacteria) with newly generated ONT direct RNA sequencing data while predicting transcripts for Listeria monocytogenes strains Scott A and RO15 (Bacteria:Firmicute), Pseudomonas aeruginosa strains SG17M and NN2 strains (Bacteria:gamma-Proteobacteria), and Haloferax volcanii (Archaea:Halobacteria) using publicly available data. From >5 million E. coli K12 ONT direct RNA sequencing reads, 2,484 mRNAs are predicted and contain more than half of the predicted E. coli proteins. While the number of predicted transcripts varied by strain based on the amount of sequence data used for the predictions, across all strains examined, the average size of the predicted mRNAs is 1.6-1.7 kbp while the median size of the predicted bacterial 5'- and 3'- UTRs are 30-90 bp. Given the lack of bacterial and archaeal transcript annotation, most predictions are of novel transcripts, but we also predicted many previously characterized mRNAs and ncRNAs, including post-transcriptionally generated transcripts and small RNAs associated with pathogenesis in the E. coli E2348/69 LEE pathogenicity islands. We predicted small transcripts in the 100-200 bp range as well as >10 kbp transcripts for all strains, with the longest transcript for two of the seven strains being the nuo operon transcript, and for another two strains it was a phage/prophage transcript. This quick, easy, inexpensive, and reproducible method will facilitate the presentation of operons, transcripts, and UTR predictions alongside CDS and protein predictions in bacterial genome annotation as important resources for the research community.

Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m5C at GCU motifs.

RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including viruses, bacteria, fungi, and animals. The algorithm consistently identified a m5C at the central position of a GCU motif. However, it also identified a m5C in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this is a frequent false prediction. In the absence of further validation, several published predictions of m5C in a GCU context should be reconsidered, including those from human coronavirus and human cerebral organoid samples.

Common Analysis of Direct RNA SequencinG CUrrently Leads to Misidentification of 5-Methylcytosine Modifications at GCU Motifs.

RNA modifications, such as méthylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including virus, bacteria, fungi, and animals. The algorithm consistently identified a 5-methylcytosine at the central position of a GCU motif. However, it also identified a 5-methylcytosine in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this a frequent false prediction. In the absence of further validation, several published predictions of 5-methylcytosine in human coronavirus and human cerebral organoid RNA in a GCU context should be reconsidered.

Msh Pilus Mutations Increase the Ability of a Free-Living Bacterium to Colonize a Piscine Host.

Symbioses between animals and bacteria are ubiquitous. To better understand these relationships, it is essential to unravel how bacteria evolve to colonize hosts. Previously, we serially passaged the free-living bacterium, Shewanella oneidensis, through the digestive tracts of germ-free larval zebrafish (Danio rerio) to uncover the evolutionary changes involved in the initiation of a novel symbiosis with a vertebrate host. After 20 passages, we discovered an adaptive missense mutation in the mshL gene of the msh pilus operon, which improved host colonization, increased swimming motility, and reduced surface adhesion. In the present study, we determined that this mutation was a loss-of-function mutation and found that it improved zebrafish colonization by augmenting S. oneidensis representation in the water column outside larvae through a reduced association with environmental surfaces. Additionally, we found that strains containing the mshL mutation were able to immigrate into host digestive tracts at higher rates per capita. However, mutant and evolved strains exhibited no evidence of a competitive advantage after colonizing hosts. Our results demonstrate that bacterial behaviors outside the host can play a dominant role in facilitating the onset of novel host associations.

Phenotypic Parallelism during Experimental Adaptation of a Free-Living Bacterium to the Zebrafish Gut.

Although animals encounter a plethora of bacterial species throughout their lives, only a subset colonize vertebrate digestive tracts, and these bacteria can profoundly influence the health and development of their animal hosts. However, our understanding of how bacteria initiate symbioses with animal hosts remains underexplored, and this process is central to the assembly and function of gut bacterial communities. Therefore, we used experimental evolution to study a free-living bacterium as it adapts to a novel vertebrate host by serially passaging replicate populations of Shewanella oneidensis through the intestines of larval zebrafish (Danio rerio). After approximately 200 bacterial generations, isolates from evolved populations improved their ability to colonize larval zebrafish during competition against their unpassaged ancestor. Genome sequencing revealed unique sets of mutations in the two evolved isolates exhibiting the highest mean competitive fitness. One isolate exhibited increased swimming motility and decreased biofilm formation compared to the ancestor, and we identified a missense mutation in the mannose-sensitive hemagglutinin pilus operon that is sufficient to increase fitness and reproduce these phenotypes. The second isolate exhibited enhanced swimming motility but unchanged biofilm formation, and here the genetic basis for adaptation is less clear. These parallel enhancements in motility and fitness resemble the behavior of a closely related Shewanella strain previously isolated from larval zebrafish and suggest phenotypic convergence with this isolate. Our results demonstrate that adaptation to the zebrafish gut is complex, with multiple evolutionary pathways capable of improving colonization, but that motility plays an important role during the onset of host association.IMPORTANCE Although animals encounter many bacterial species throughout their lives, only a subset colonize vertebrate digestive tracts, and these bacteria can profoundly influence the health and development of their animal hosts. We used experimental evolution to study a free-living bacterium as it adapts to a novel vertebrate host by serially passaging replicate populations of Shewanella oneidensis through the intestines of larval zebrafish (Danio rerio). Our results demonstrate that adaptation to the zebrafish gut is complex, with multiple evolutionary pathways capable of improving colonization, but that motility plays an important role during the onset of host association.

Complete Genome Sequence of wBp, the Wolbachia Endosymbiont of Brugia pahangi FR3.

Lymphatic filariasis is a devastating disease caused by filarial nematode roundworms, which contain obligate Wolbachia endosymbionts. Here, we assembled the genome of wBp, the Wolbachia endosymbiont of the filarial nematode Brugia pahangi, from Illumina, Pacific Biosciences, and Oxford Nanopore data. The complete, circular genome is 1,072,967 bp.

Progression of asymptomatic carotid stenosis: a natural history study in 1004 patients.

PURPOSE: The purpose of this study was to delineate the natural history of the progression of asymptomatic carotid stenosis. METHODS: In a 10-year period, 1701 carotid arteries in 1004 patients who were asymptomatic were studied with serial duplex scans (mean follow-up period, 28 months; mean number of scans, 2.9/patient). At each visit, stenoses of the internal carotid artery (ICA) and the external carotid artery (ECA) were categorized as none (0 to 14%), mild (15% to 49%), moderate (50% to 79%), severe (80% to 99%), preocclusive, or occluded. Progression was defined as an increase in ICA stenosis to >/=50% for carotid arteries with a baseline of <50% or as an increase to a higher category of stenosis if the baseline stenosis was >/=50%. The Cox proportional hazards model was used for data analysis. RESULTS: The risk of progression of ICA stenosis increased steadily with time (annualized risk of progression, 9.3%). With multivariate modeling, the four most important variables that affected the progression (P <.02) were baseline ipsilateral ICA stenosis >/=50% (relative risk [RR], 3.34), baseline ipsilateral ECA stenosis >/=50% (RR, 1.51), baseline contralateral ICA stenosis >/=50% (RR, 1.41), and systolic pressure more than 160 mm Hg (RR, 1. 37). Ipsilateral neurologic ischemic events (stroke/transient ischemic attack) occurred in association with 14.0% of the carotid arteries that were studied. The progression of ICA stenosis correlated with these events (P <.001), but baseline ICA stenosis was not a significant predictor. CONCLUSION: In contrast to recently published studies, we found that the risk of progression of carotid stenosis is substantial and increases steadily with time. Baseline ICA stenosis was the most important predictor of the progression, but baseline ECA stenosis also was identified as an important independent predictor. Contralateral ICA stenosis and systolic hypertension were additional significant predictors. We found further that the progression of ICA stenosis correlated with ischemic neurologic events but not baseline stenosis. The data provide justification for the use of serial duplex scans to follow carotid stenosis and suggest that different follow-up intervals may be appropriate for different patient subgroups.

Long-term results of femoropopliteal bypass with stabilized human umbilical vein.

BACKGROUND: Stabilized human umbilical vein (SHUV) is one of several graft materials that may be used when autogenous saphenous vein is absent or inadequate, or when a shortened anesthesia time is deemed necessary. METHODS: Two hundred eleven consecutive femoropopliteal bypasses were used in 171 patients since 1977. Follow-up has been conducted at regular intervals since operation, and the results scrutinized according to operative indication, diabetic status, and the number of runoff vessels. RESULTS: Life-table cumulative rates were 70 +/- 3%, 45 +/- 4%, and 26 +/- 5% at 1, 5, and 10 years, respectively. Patency rates for bypasses performed for claudication were superior to those performed for limb-salvage but achieved statistical significance at 1 and 3 years only. Early patency rates for nondiabetic patients were superior to those in patients with diabetes, but did not achieve statistical significance. The incidence of infection was 3%, and the incidence of aneurysm formation was 3.3%. CONCLUSION: SHUV is an acceptable alternative for femoropopliteal bypass when autogenous saphenous vein is inadequate or unavailable.

Cavernous hemangioma of the cephalic vein.

Although capillary and venous hemangiomas are common problems in children, cavernous hemangiomas of extremity veins in adults are unusual. Successful operative removal of a cavernous hemangioma in the right forearm of a 37-year-old man is described. To the author's knowledge, such lesions in named upper-extremity veins have not previously been reported.

Anastomotic suture materials and experimental colorectal carcinogenesis.

Local tumor recurrence following restorative surgery for colorectal cancer may occasionally result from the promotion of a neoplastic lesion in a zone of proliferative instability adjacent to the anastomosis. The aim of this study was to compare the influence of three anastomotic suture materials, including stainless steel (as a model of surgical stapling), on colorectal carcinogenesis in an experimental animal model. The transmural implantation of stainless steel sutures into the distal descending colon of albino Swiss rats during the postinitiation phase of tumor induction resulted in significantly fewer animals exhibiting perianastomotic tumors 12 weeks later (3 of 21 animals) when compared with either polyamide (Nurolon; Ethicon, Edinburgh, United Kingdom) (14 of 20 animals; P less than 0.001) or polyglycolic acid (Dexon Plus; Davis and Geck, Gosport, United Kingdom) sutures (17 of 21 animals; P less than 0.001). The findings were similar when the same materials were used to resuture a longitudinal colotomy. For both operative procedures, the type of suture material had no influence on the incidence of large bowel tumors distant from the anastomotic site. These results suggest that stainless steel staples may promote fewer perianastomotic large bowel tumors than certain more conventional suture materials and, therefore, may be safely employed in colorectal cancer surgery.

Reoperation for complications of stabilized human umbilical vein grafts.

Among 211 femoropopliteal bypass grafts constructed with stabilized human umbilical vein performed between 1977 and 1986, 3 major complications were seen: graft thrombosis, aneurysm formation, and infection. Early thrombosis (within 30 days of operation) was successfully treated in 14 of 17 patients (82 percent) with either graft thrombectomy (13 of 17 patients) or graft replacement (1 patient). Late graft closure (2 to 55 months after operation) was successfully treated with thrombectomy in 17 of 28 patients (61 percent) or graft revision or replacement in 4 patients (14 percent). Aneurysms and graft infections were each seen in 3 percent of the grafts at risk. The former were successfully reconstructed in six of seven instances, whereas the latter eventuated in amputation in six of seven patients. An aggressive approach to graft closure in stabilized human umbilical vein grafts is recommended as this conduit is amenable to thrombectomy or revision if closure occurs.

Inflammatory aneurysms of the abdominal aorta.

Inflammatory aneurysms of the abdominal aorta (IAAAs) have distinctive clinical and physical characteristics that separate them from typical atherosclerotic aneurysms. They were identified in 19 (7.2%) of 265 patients undergoing abdominal aortic aneurysm repair. Symptoms were present in 12 (63%) of 19, with one patient presenting with rupture, and multiple symptoms were present in six (32%). Intraoperatively, all aneurysms exhibited dense periaortic inflammation. Adjacent structures most frequently involved were the duodenum in 15 (79%) of 19 patients, the left renal vein in six (32%) of 19, and the ureter in five (26%) of 19. Seventeen (94%) of the 18 patients who underwent elective aneurysm resection survived. The involvement of retroperitoneal structures varied in number and severity, demonstrating that a wide spectrum of inflammation is present in IAAAs, making diagnosis and definition difficult.

Dietary manipulation during experimental colorectal carcinogenesis: a morphological study in the rat.

The effect of the dietary manipulation of fat and fibre on the gross pathological, histopathological and scanning electron microscopic appearances of colonic mucosa was studied before and during experimental carcinogenesis in 232 male Albino Swiss rats. Carcinogen treated animals were given 12 consecutive weekly injections of Azoxymethane in a dose of 10 mg/kg per week. The animals were divided between four dietary groups (1: high fat high fibre, 2: low fat high fibre, 3: high fat low fibre and 4: low fat low fibre). The colorectal morphology was assessed at autopsy using standard histopathological techniques. In addition, scanning electron microscopic studies on selected samples were performed. A scoring system was introduced to allow a more accurate comparison of the surface architectural appearances. The high fat low fibre diet was associated with the greatest risk for macroscopic tumour production and the low fat high fibre diet with the lowest risk. Statistically significant differences between each of the dietary groups were noted with the exception of the comparison for tumour induction between the high fat high fibre and low fat high fibre dietary groups. Histopathological analysis confirmed the inter dietary relationships with respect to the extent of neoplastic change. The scanning electron microscopic study revealed that both dietary manipulation and carcinogen treatment influenced the surface characteristics. Control animals fed diet 2 (low fat high fibre) but not treated with carcinogen, showed the greatest deviation from the normal appearances. Animals treated with carcinogen and fed diet 3 (high fat low fibre) consistently showed the greatest surface abnormalities.

Morphological and cell kinetic effects of dietary manipulation during colorectal carcinogenesis.

The effect of dietary manipulation of fat and fibre on the structural and cell kinetic characteristics of colonic mucosa was studied before and during experimental carcinogenesis in 232 male Albino Swiss rats. Carcinogen treated animals were given 12 weekly injections of azoxymethane (10 mg/kg/week). The animals were divided between four dietary groups (1) high fat, high fibre, (2) low fat, high fibre, (3) high fat, low fibre and (4) low fat, low fibre. Pathological and cell kinetic information together with details of certain faecal characteristics was collected when the animals were killed 4, 20, and 28 weeks after starting their experimental diet. Tumour induction was significantly influenced by diet. The highest risk of colorectal tumour development was found in groups fed diet 3: high fat, low fibre (p less than 0.03). In contrast, diet 2: low fat, high fibre was associated with the lowest risk. The proportion of histologically proven colonic tumours occurring in each dietary group was: diet 1-10.9%, diet 2-3.6%, diet 3-63.7%, diet 4-21.8%. Scanning electron microscopic (SEM) studies done on selected samples indicated both dietary and azoxymethane related alterations in crypt unit integrity. The most marked surface architectural changes were seen in carcinogen treated animals maintained on diet 3 (high fat, low fibre). Stathmokinetic analysis revealed considerable intergroup variability. Both fat and fibre produced significant effects, principally during the preneoplastic phase of carcinogenesis. Faster proliferative activity tended to be found in animals at low risk of tumour induction (diet 2), slower proliferation being more characteristic of animals at high risk (p less than 0.05). The findings suggest that both topographical and cell kinetic parameters have an important relationship with promoting and protecting dietary factors during the development of colorectal cancer.

Experimental colorectal cancer: the relationship of diet and faecal bile acid concentration to tumour induction.

Epidemiological studies have consistently suggested an aetiological relationship between certain dietary constituents, faecal bile acid (FBA) concentration and colorectal cancer. This study was designed to examine the effect of the dietary manipulation of fat and fibre on tumour induction and on various faecal characteristics in Albino Swiss rats. A total of 232 animals were maintained on one of four different diets for 4, 20 and 28 weeks respectively. The diets were classified as high fat high fibre, low fat high fibre, high fat low fibre and low fat low fibre. The groups were further sub-divided according to the administration of systemic azoxymethane (10 mg/kg per week) or saline over 12 consecutive weeks. The high fat low fibre diet was associated with the highest risk for tumour production and the low fat high fibre diet with the lowest risk. Statistically significant differences between all the diets were noted with the exception of a comparison between the high fat high fibre and low fat high fibre diets. The highest total concentration of free FBA was found in the faeces from animals fed low fibre containing diets. The results show a clear influence of both fat and fibre on tumour induction while, in this model, fibre was the principle determinant of faecal bile acid concentration.