Expression in CHO cells of a bacterial biosynthetic pathway producing a small non-ribosomal peptide aldehyde prevents proteolysis of recombinant proteins.

A significant problem during recombinant protein production is proteolysis. One of the most common preventive strategies is the addition of protease inhibitors, which has drawbacks, such as their short half-life and high cost, and their limited prevention of extracellular proteolysis. Actinomycetes produce the most commonly used inhibitors, which are non-ribosomal small aldehydic peptides. Previously, an unprecedented biosynthetic route involving a condensation-minus non-ribosomal peptide synthetase (NRPSs) and a tRNA utilizing enzyme (tRUE) was shown to direct the synthesis of one of these inhibitor peptides, livipeptin. Here, we show that expression of the livipeptin biosynthetic pathway encoded by the lvp genes in CHO cells resulted in the production of this metabolite with cysteine protease inhibitory activity, implying that mammalian tRNAs were recruited by the lvp system. CHO cells transiently expressing the biosynthetic pathway produced livipeptin without affecting cell growth or viability. Expression of the lvp system in CHO cells producing two model proteins, secreted alkaline phosphatase (hSeAP) and a monoclonal antibody, resulted in higher specific productivity with reduced proteolysis. We show for the first time that the expression of a bacterial biosynthetic pathway is functional in CHO cells, resulting in the efficient, low-cost synthesis of a protease inhibitor without adverse effects on CHO cells. This expands the field of metabolic engineering of mammalian cells by expressing the overwhelming diversity of actinomycetes biosynthetic pathways and opens a new option for proteolysis inhibition in bioprocess engineering.

PhyloString: A web server designed to identify, visualize, and evaluate functional relationships between orthologous protein groups across different phylogenetic lineages.

Proteins are biological units whose essence is defined by their functional relationships with other proteins or biomolecules such as RNA, DNA, lipids, or carbohydrates. These functions encompass enzymatic, structural, regulatory, or physical interaction roles. The STRING database (Nucleic Acids Research, 8 Jan 2021;49(D1): D605-12) provides an index that defines the functional interaction networks between proteins in model organisms. To facilitate the identification, visualization, and evaluation of potential functional networks across organisms from different phylogenetic lineages, we have developed PhyloString (https://biocomputo.ibt.unam.mx/phylostring/), a web server that utilizes the indices of the STRING database. PhyloString decomposes these functional networks into modules, representing cohesive units of proteins grouped based on their similarity of STRING values and the phylogenetic origins of their respective organisms. This study presents and thoroughly discusses examples of such functional networks and their modules identified using PhyloString.

Single metachronous bone metastasis following rectal adenocarcinoma: A case report.

Colorectal cancer (CRC) ranks as the third leading cause of cancer-related mortality in developed countries. While its incidence in early stages has increased due to screening programs, a significant number of patients experience the development of metastases either at the time of diagnosis or during follow-ups. Unlike certain other types of cancer, such as breast, prostate, or lung cancer, where bone tissue is a common site for secondary dissemination, CRC primarily spreads to the lymph nodes, liver and lungs. The occurrence of bone metastases from CRC is rare and usually coincides with tumor involvement in other locations. Risk factors for bone metastases include the location of the primary tumor, the age of the patients, KRAS mutations and the degree of tumor differentiation. Unlike metastases to the liver and lungs, bone metastases tend to be symptomatic, affecting the patient's quality of life and resulting in a poorer prognosis with shorter survival rates. The approach to patient management needs to be personalized. The present study describes the of a patient who underwent surgery for stage IV rectal adenocarcinoma and later developed a metastasis in the costal wall 79 months post-intervention, with no evidence of recurrence at other sites.

Unraveling the plasticity of translation initiation in prokaryotes: Beyond the invariant Shine-Dalgarno sequence.

Translation initiation in prokaryotes is mainly defined, although not exclusively, by the interaction between the anti-Shine-Dalgarno sequence (antiSD), located at the 3'-terminus of the 16S ribosomal RNA, and a complementary sequence, the ribosome binding site, or Shine-Dalgarno (SD), located upstream of the start codon in prokaryotic mRNAs. The antiSD has a conserved 5'-CCUCC-3' core, but inter-species variations have been found regarding the participation of flanking bases in binding. These variations have been described for certain bacteria and, to a lesser extent, for some archaea. To further analyze these variations, we conducted binding-energy prediction analyses on over 6,400 genomic sequences from both domains. We identified 15 groups of antiSD variants that could be associated with the organisms' phylogenetic origin. Additionally, our findings revealed that certain organisms exhibit variations in the core itself. Importantly, an unaltered core is not necessarily required for the interaction between the 3'-terminus of the rRNA and the region preceding the AUG of the mRNA. In our study, we classified organisms into four distinct categories: i) those possessing a conserved core and demonstrating binding; ii) those with a conserved core but lacking evidence of binding; iii) those exhibiting binding in the absence of a conserved core; and iv) those lacking both a conserved core and evidence of binding. Our results demonstrate the flexibility of organisms in evolving different sequences involved in translation initiation beyond the traditional Shine-Dalgarno sequence. These findings are discussed in terms of the evolution of translation initiation in prokaryotic organisms.

Spontaneous cervical hematoma caused by giant parathyroid adenoma in an elderly patient: A case report.

Spontaneous cervical hematoma usually occurs as a consequence of extracapsular bleeding from a parathyroid gland, generally due to the presence of an adenoma (giant adenoma), glandular hyperplasia, cystic component, or, less frequently, due to the existence of a carcinoma. The hematoma can be confined to the cervical compartment or extend to the mediastinum, potentially causing airway compression. Despite this, the recommended management in hemodynamically stable patients consists of surveillance and hospital monitoring with delayed surgery after a few weeks. On the other hand, in those patients with airway compromise and instability, emergency surgery, consisting of cervicotomy and drainage, is mandatory. The present study describes the case of a 78-year-old patient with a medical history of high blood pressure, non-insulin-dependent diabetes mellitus, dyslipidemia, moderate aortic stenosis, chronic kidney disease and sarcoidosis under pharmacological treatment who attended the emergency department due to symptoms of neck pain, an increase in soft tissue, and dyspnea on moderate exertion with an evolution leading to respiratory failure. This was secondary to a diagnosis of spontaneous cervical hematoma that required urgent surgical intervention. The results of histopathological analysis revealed that a giant parathyroid adenoma was responsible for the bleeding. The patient had a complicated post-operative period with a prolonged admission to the intensive care unit.

The Azotobacter vinelandii AlgU regulon during vegetative growth and encysting conditions: A proteomic approach.

In the Pseduomonadacea family, the extracytoplasmic function sigma factor AlgU is crucial to withstand adverse conditions. Azotobacter vinelandii, a closed relative of Pseudomonas aeruginosa, has been a model for cellular differentiation in Gram-negative bacteria since it forms desiccation-resistant cysts. Previous work demonstrated the essential role of AlgU to withstand oxidative stress and on A. vinelandii differentiation, particularly for the positive control of alginate production. In this study, the AlgU regulon was dissected by a proteomic approach under vegetative growing conditions and upon encystment induction. Our results revealed several molecular targets that explained the requirement of this sigma factor during oxidative stress and extended its role in alginate production. Furthermore, we demonstrate that AlgU was necessary to produce alkyl resorcinols, a type of aromatic lipids that conform the cell membrane of the differentiated cell. AlgU was also found to positively regulate stress resistance proteins such as OsmC, LEA-1, or proteins involved in trehalose synthesis. A position-specific scoring-matrix (PSSM) was generated based on the consensus sequence recognized by AlgU in P. aeruginosa, which allowed the identification of direct AlgU targets in the A. vinelandii genome. This work further expands our knowledge about the function of the ECF sigma factor AlgU in A. vinelandii and contributes to explains its key regulatory role under adverse conditions.

Essential gene complement of Planctopirus limnophila from the bacterial phylum Planctomycetes.

Planctopirus limnophila belongs to the bacterial phylum Planctomycetes, a relatively understudied lineage with remarkable cell biology features. Here, we report a genome-wide analysis of essential gene content in P. limnophila. We show that certain genes involved in peptidoglycan synthesis or cell division, which are essential in most other studied bacteria, are not essential for growth under laboratory conditions in this species. We identify essential genes likely involved in lipopolysaccharide biosynthesis, consistent with the view of Planctomycetes as diderm bacteria, and highlight other essential genes of unknown functions. Furthermore, we explore potential stages of evolution of the essential gene repertoire in Planctomycetes and the related phyla Verrucomicrobia and Chlamydiae. Our results provide insights into the divergent molecular and cellular biology of Planctomycetes.

The effect of the genomic GC content bias of prokaryotic organisms on the secondary structures of their proteins.

One of the main characteristics of prokaryotic genomes is the ratio in which guanine-cytosine bases are used in their DNA sequences. This is known as the genomic GC content and varies widely, from values below 20% to values greater than 74%. It has been demonstrated that the genomic GC content varies in accordance with the phylogenetic distribution of organisms and influences the amino acid composition of their corresponding proteomes. This bias is particularly important for amino acids that are coded by GC content-rich codons such as alanine, glycine, and proline, as well as amino acids that are coded by AT-rich codons, such as lysine, asparagine, and isoleucine. In our study, we extend these results by considering the effect of the genomic GC content on the secondary structure of proteins. On a set of 192 representative prokaryotic genomes and proteome sequences, we identified through a bioinformatic study that the composition of the secondary structures of the proteomes varies in relation to the genomic GC content; random coils increase as the genomic GC content increases, while alpha-helices and beta-sheets present an inverse relationship. In addition, we found that the tendency of an amino acid to form part of a secondary structure of proteins is not ubiquitous, as previously expected, but varies according to the genomic GC content. Finally, we discovered that for some specific groups of orthologous proteins, the GC content of genes biases the composition of secondary structures of the proteins for which they code.

Malignant adipocytic tumours: A 20­year single­centre retrospective study.

Adipocytic tumours are the most common soft tissue neoplasms. Among them, liposarcoma is the most frequent malignant neoplasm. However, to the best of our knowledge, no previously published study has assessed the evolution and oncological prognosis of the different subtypes of liposarcoma at the retroperitoneal level compared with at other locations. The present study is a retrospective observational study in which all patients were operated on between October 2000 and January 2020 with a histological diagnosis of liposarcoma. Variables, such as age, sex, location, histological type, recurrence, type of treatment and mortality, among others, were analysed. The patients were divided into two groups: Group A (retroperitoneal location) and group B (non-retroperitoneal location). A total of 52 patients with a diagnosis of liposarcoma (17 women and 35 men) and a mean age of 57.2±15.9 years were assessed. A total of 16 patients were classified into group A and 36 into group B. The OR of recurrence was 1.5 (P=0.02) for R1 vs. R0 resection in group A. The OR of recurrence in group B for R1 vs. R0 resection was 1.8 (P=0.77), whereas for R2 vs. R0 resection, the OR was 69 (P=0.011). In conclusion, 52 cases of malignant adipocytic tumours collected during 2000-2020 were analysed with the new World Health Organization classification (updated 2020). Although its recurrence potential and capacity for distant metastasis depended on each histological type, surgical treatment with unaffected margins was the main prognostic factor for survival. The present study identified differences in relation to the survival of each histological subtype and its location, finding greater survival in dedifferentiated liposarcoma, myxoid liposarcoma and pleomorphic liposarcoma located at the extraperitoneal level than in the retroperitoneal location. Resectability was not influenced by liposarcoma location.

Antisense-acting riboswitches: A poorly characterized yet important model of transcriptional regulation in prokaryotic organisms.

Riboswitches are RNA elements involved in regulating genes that participate in the biosynthesis or transport of essential metabolites. They are characterized by their ability to recognize their target molecules with high affinity and specificity. Riboswitches are commonly cotranscribed with their target genes and are located at the 5' end of their transcriptional units. To date, only two exceptional cases of riboswitches being situated at the 3' end and transcribing in the antisense direction of their regulated genes have been described. The first case involves a SAM riboswitch located at the 3' end of the ubiG-mccB-mccA operon in Clostridium acetobutylicum involved in converting methionine to cysteine. The second case concerns a Cobalamin riboswitch in Listeria monocytogenes that regulates the transcription factor PocR related to this organism's pathogenic process. In almost a decade since the first descriptions of antisense-acting riboswitches, no new examples have been described. In this work, we performed a computational analysis to identify new examples of antisense-acting riboswitches. We found 292 cases in which, according to the available information, we infer that the expected regulation of the riboswitch is consistent with the signaling molecule it senses and the metabolic function of the regulated gene. The metabolic implications of this novel type of regulation are thoroughly discussed.

Hyperthermic Intraperitoneal Chemotherapy and Recirculation with CO2: A Safe Technique.

INTRODUCTION: Hyperthermic IntraPEritoneal Chemotherapy (HIPEC) has evolved as a treatment for peritoneal carcinomatosis in various tumors after a careful and complete cytoreductive surgery, and it demonstrated much better and longer survival than more traditional therapeutic schemas. Our objective has been to examine the safety, efficacy and survival achieved with closed technique with CO2-agitation system Combat PRS® (Peritoneal Recirculation System: PRS). To achieve this, we compared the appearance of adverse events, mortality and survival with the described using classic techniques (open, closed without CO2-agitation) for the treatment of selected patients with peritoneal carcinomatosis; Materials and methods: We studied overall survival, disease-free survival and safety (morbidity and mortality) of the administration of HIPEC through a closed method technique with CO2 recirculation (Combat PRS®) in 482 patients from 11 Spanish hospitals; Results: The mortality of our technique (1.66%) was similar to other published techniques (open, closed). Morbidity exhibited a 9.96% rate of Clavien-Dindo (CD) III/IV complications in 482 patients, which was lower than in other series. Survival (overall survival (OS) and disease-free survival (DFS)) was similar to previously published results: 86% 1y-OS, 54% 3y-OS, 77% 1y-DFS and 31% 3y-DFS; Conclusion: The procedure with closed PRS with CO2 agitation is as safe as standard open and closed procedures for the administration of HIPEC after complete cytoreductive surgery, with similar and very low mortality (1.66%) and lower morbidity (9.96% CD III and IV in our series vs range of 20-40% in the majority of different series); only Kusamura had similar results, with 12% in 205 patients, using the closed technique without CO2 agitation).

Prognostic Factors of Survival in Patients with Peritoneal Metastasis from Colorectal Cancer.

Objectives: The aim of this study was to analyze the prognostic factors of survival in patients with peritoneal metastasis (PM) from colorectal cancer (CRC). The type of relationship between survival and the PM time of detection was used to determine whether it was synchronous with the primary tumor or metachronous. Patients and Methods: Retrospective observational study. It included patients treated for colorectal adenocarcinoma diagnosed between January 2005 and December 2019 who presented PM at the time of diagnosis or during follow-up. Variables, such as sex, age, differentiation grade, positive adenopathy (pN+), tumor size (pT), tumor location, mucinous component, peritoneal carcinomatosis index (PCI), and KRAS mutational status, were analyzed. Results: During the study period, 1882 patients were surgically treated for CRC in our hospital. Of these, 240 patients (12.8%) were included in the study after evidence of PM. The mean age was 67 ± 12 years (range: 32−92 years), and 114 patients were female (47.5%). The mean follow-up was 20 ± 13 months (median 12 months). The Kaplan−Meier survival at 36 months was higher in patients with metachronous PM (24% vs. 8%; p = 0.002), WT-KRAS tumors (31% vs. 15%; p < 0.001), N0 stage (30% vs. 19%; p < 0.001), T3 stage tumors (18% vs. 19% in T4A and 3% in T4B; p > 0.001), and tumors with classic adenocarcinoma histology (18% vs. 8%; p = 0.011). Patients with a PCI of 1−10 showed a likelihood of survival at 36 months of 56%, which was longer than that found in patients with a PCI of 11−20 (8%) or a PCI of >20 (0%) (p < 0.001). In the multiple regression analysis, the factors with an independent prognostic value were: poor grade of differentiation (HR 1.995; 95% CI: 1.294−3.077), KRAS mutation (HR 1.751; 95% CI: 1.188−2.581), PCI 11−20 (HR: 9.935; 95% CI: 5.204−18.966) and PCI > 20 (HR: 4.011; 95% CI: 2.291−7.023). Conclusions: PCI should continue as the as the most useful prognostic indicator in order to assess prognostic estimations as well as therapeutic and surgical decisions, but tumor grade and KRAS mutational status may help in the treatment decision process by providing complementary information. The time of PM detection did not achieve statistical significance in the multiple regression analysis.

Prognostic Value of KRAS Gene Mutation on Survival of Patients with Peritoneal Metastases of Colorectal Adenocarcinoma.

OBJECTIVE: The main objective of the study was to determine the effect of the presence of mutation in the KRAS gene on the survival in patients with colorectal cancer (CRC) and peritoneal metastases (PM). MATERIALS AND METHODS: A retrospective cohort study was performed. Patients diagnosed with CRC with synchronous or metachronous PM between January 2006 and December 2019 were included. Data on the histopathological, clinical, and treatment factors were collected. The effect of each variable on survival was evaluated by Cox regression. RESULTS: A total of 149 patients were included (64 women (43%) and 85 men (57%); mean age, 63 years). The long-term survival rate at 36 months was 24% (median, 21 months). KRAS mutation was detected in 75 patients (50.3%). Kaplan-Meier analysis estimated that likelihood of survival was higher in patients with wild-type KRAS tumours (35%) than in mutated-type KRAS (14%) (median: 28 vs. 15, respectively) (P=0.001). Within the categories into which the peritoneal cancer index (PCI) was classified, survival at 36 months depended on the KRAS status. Survival in wild-type KRAS tumours with PCI 1-10 was 71% and with PCI 11-20 was 26%, while in mutant-type KRAS tumours, survival was 41% and 4%, respectively (P=0.025). In the multiple regression analysis, the KRAS mutation was revealed to have an independent prognostic value (HR: 2.144; 95% CI: 1.342-3.424). CONCLUSION: The mutational status of the KRAS gene has demonstrated a strong association with survival and prognostic utility in patients with CRC with PM.

Bilateral Nipple Enlargement as a Secondary Effect of Anabolic Drugs: A Histopathological Mimicker of Smooth Muscle Hamartoma.

Smooth muscle hamartoma are usually solitary and congenital, may affect the genital area and nipples. Histopathologically, they are characterized by the presence of mature smooth muscle bundles. We present a 40 year-old male with bilateral nipple enlargement excised with clinical suspicion of bilateral leiomyoma. Skin biopsy shows mature, irregularly arranged smooth muscle bundles and lactiferous ducts between them. Immunohistochemistry is positive for smooth muscle actin, desmin and fumarase, but negative for estrogen and progestogen receptors. The presence of lactiferous ducts excludes bilateral leiomyomas. Even when, histopathologically, this can be interpreted as the nipple-type of muscular hamartoma of the breast, clinical history favors an anabolic drug-induced lesion. Bodybuilders present gynecomastia and nipple enlargement as frequent problems, but we have not found any histopathological description of these nipple lesions. We consider that dermatologists should be aware of the presence of them and dermatopathologists should know their histopathological features to avoid misdiagnosis as neoplasms.

Unusual Case of Gallbladder Adenocarcinoma Metastasis to the Abdominal Wall 11 Years Later: Synchronous Presentation with Two Malignant Colon Tumors, Coincidence or Not?

INTRODUCTION: Abdominal wall masses are a common finding in clinical practice. A high percentage of these masses are malignant. We present the case of a patient operated for a gallbladder adenocarcinoma, who consulted eleven years later for a malignant mass of the abdominal wall in synchrony with two adenocarcinomas of the left colon and sigmoid. Case Report. A 75-year-old male underwent a laparoscopic cholecystectomy with an incidental diagnosis of adenocarcinoma in situ (TisN0M0 according to AJCC 8th edition). The operative report mentioned that the removal of the gallbladder was difficult due to the inflammatory process, and the gallbladder was accidentally opened during the operation. It was not clear from the operative report whether an extraction bag was utilized to remove the specimen, but the histopathological study confirmed an open gallbladder. He presented 11 years later with an asymptomatic heterogeneous complex cystic mass involving the anterior rectus abdominis muscle. Colonoscopy showed synchronous tumors in the descending and sigmoid colon with pathology confirming adenocarcinoma. The patient underwent an elective laparotomy with resection of the anterior abdominal wall mass, left hemicolectomy, and sigmoidectomy. The histopathological results of the abdominal mass (CK7, CK20, EMA, CEA positive) were described as metastasis of adenocarcinoma of biliary origin. Discussion. Port site recurrences are rare complications following laparoscopic surgery when malignancy is unsuspected. Possible factors related to local implantation include direct seeding of spilled bile or tumor cells into the wound or shedding of tumor cells due to pneumoperitoneum-induced loss of the peritoneal barrier at the trocar site. In the absence of distant metastasis, treatment should include wide port site excision with malignancy-free surgical margins. CONCLUSION: Abdominal wall metastasis from gallbladder carcinoma is rare, and its synchronous presentation with a malignant neoplasm of the colon is exceptional. This is the first report of a patient with abdominal wall metastasis from a gallbladder adenocarcinoma operated eleven years ago that debuted synchronously with two adenocarcinomas of the left colon and sigma.

Definition of the Metagenomic Profile of Ocean Water Samples From the Gulf of Mexico Based on Comparison With Reference Samples From Sites Worldwide.

Computational and statistical analysis of shotgun metagenomes can predict gene abundance and is helpful for elucidating the functional and taxonomic compositions of environmental samples. Gene products are compared against physicochemical conditions or perturbations to shed light on the functions performed by the microbial community of an environmental sample; however, this information is not always available. The present study proposes a method for inferring the metabolic potential of metagenome samples by constructing a reference based on determining the probability distribution of the counts of each enzyme annotated. To test the methodology, we used marine water samples distributed worldwide as references. Then, the references were utilized to compare the annotated enzymes of two different water samples extracted from the Gulf of Mexico (GoM) to distinguish those enzymes with atypical behavior. The enzymes whose annotation counts presented frequencies significantly different from those of the reference were used to perform metabolic reconstruction, which naturally identified pathways. We found that several of the enzymes were involved in the biodegradation of petroleum, which is consistent with the impact of human hydrocarbon extraction activity and its ubiquitous presence in the GoM. The examination of other reconstructed pathways revealed significant enzymes indicating the presence of microbial communities characterizing each ocean depth and ocean cycle, providing a fingerprint of each sampled site.