Biodegradation of selected aminophosphonates by the bacterial isolate Ochrobactrum sp. BTU1.

Aminophosphonates, like glyphosate (GS) or metal chelators such as ethylenediaminetetra(methylenephosphonic acid) (EDTMP), are released on a large scale worldwide. Here, we have characterized a bacterial strain capable of degrading synthetic aminophosphonates. The strain was isolated from LC/MS standard solution. Genome sequencing indicated that the strain belongs to the genus Ochrobactrum. Whole-genome classification using pyANI software to compute a pairwise ANI and other metrics between Brucella assemblies and Ochrobactrum contigs revealed that the bacterial strain is designated as Ochrobactrum sp. BTU1. Degradation batch tests with Ochrobactrum sp. BTU1 and the selected aminophosphonates GS, EDTMP, aminomethylphosphonic acid (AMPA), iminodi(methylene-phosphonic) (IDMP) and ethylaminobis(methylenephosphonic) acid (EABMP) showed that the strain can use all phosphonates as sole phosphorus source during phosphorus starvation. The highest growth rate was achieved with AMPA, while EDTMP and GS were least supportive for growth. Proteome analysis revealed that GS degradation is promoted by C-P lyase via the sarcosine pathway, i.e., initial cleavage at the C-P bond. We also identified C-P lyase to be responsible for degradation of EDTMP, EABMP, IDMP and AMPA. However, the identification of the metabolite ethylenediaminetri(methylenephosphonic acid) via LC/MS analysis in the test medium during EDTMP degradation indicates a different initial cleavage step as compared to GS. For EDTMP, it is evident that the initial cleavage occurs at the C-N bond. The detection of different key enzymes at regulated levels, form the bacterial proteoms during EDTMP exposure, further supports this finding. This study illustrates that widely used and structurally more complex aminophosphonates can be degraded by Ochrobactrum sp. BTU1 via the well-known degradation pathways but with different initial cleavage strategy compared to GS.

Synergic Induction of Autophagic Cell Death in Anaplastic Thyroid Carcinoma.

Anaplastic thyroid carcinoma (ATC) has poor prognosis, high mortality rate and lack of effective therapy. A synergic combination of PD-L1 antibody together with cell death promoting substances like deacetylase inhibitors (DACi) and multi-kinase inhibitors (MKI) could sensitize ATC cells and promote decay by autophagic cell death. The PD-L1-inhibitor atezolizumab synergized with panobinostat (DACi) and sorafenib (MKI) leading to significant reduction of the viability, measured by real time luminescence, of three different patient-derived primary ATC cells, of C643 cells and follicular epithelial thyroid cells too. Solo administration of these compounds caused a significant over-expression of autophagy transcripts; meanwhile autophagy proteins were almost not detectable after the single administration of panobinostat, thus supporting a massive autophagy degradation process. Instead, the administration of atezolizumab caused an accumulation of autophagy proteins and the cleavage of the active caspases 8 and 3. Interestingly, only panobinostat and atezolizumab were able to exacerbate the autophagy process by increasing the synthesis, the maturation and final fusion with the lysosomes of the autophagosome vesicles. Despite ATC cells could be sensitized by atezolizumab via the cleavage of the caspases, no reduction of cell proliferation or promotion of cell death was observed. The apoptosis assay evidenced the ability of panobinostat alone and in combination with atezolizumab to induce the phosphatidil serine exposure (early apoptosis) and further the secondary necrosis. Instead, sorafenib was only able to cause necrosis. The increase of caspases activity induced by atezolizumab, the apoptosis and autophagy processes promoted by panobinostat synergize thus promoting cell death in well-established and primary anaplastic thyroid cancer cells. The combined therapy could represent a future clinical application for the treatment of such lethal and untreatable solid cancer.

Tracking the distribution, genetic diversity and lineage of Brucella melitensis recovered from humans and animals in Egypt based on core-genome SNP analysis and in silico MLVA-16.

Brucellosis is one of the most common neglected zoonotic diseases globally, with a public health significance and a high economic loss in the livestock industry caused by the bacteria of the genus Brucella. In this study, 136 Egyptian Brucella melitensis strains isolated from animals and humans between 2001 and 2020 were analysed by examining the whole-core-genome single-nucleotide polymorphism (cgSNP) in comparison to the in silico multilocus variable number of tandem repeat analysis (MLVA-16). Almost all Egyptian isolates were belonging to the West Mediterranean clade, except two isolates from buffalo and camel were belonging to the American and East Mediterranean clades, respectively. A significant correlation between the human case of brucellosis and the possible source of infection from animals was found. It seems that several outbreak strains already existing for many years have been spread over long distances and between many governorates. The cgSNP analysis, in combination with epidemiological metadata, allows a better differentiation than the MLVA-16 genotyping method and, hence, the source definition and tracking of outbreak strains. The MLVA based on the currently used 16 markers is not suitable for this task. Our results revealed 99 different cgSNP genotypes with many different outbreak strains, both older and widely distributed ones and rather newly introduced ones as well. This indicates several different incidents and sources of infections, probably by imported animals from other countries to Egypt. Comparing our panel of isolates to public databases by cgSNP analysis, the results revealed near relatives from Italy. Moreover, near relatives from the United States, France, Austria and India were found by in silico MLVA.

Vessel-Sparing Lymphadenectomy Should Be Performed in Small Intestine Neuroendocrine Neoplasms.

INTRODUCTION: The goal of primary tumor resection with lymphadenectomy (PTR) in small intestine neuroendocrine neoplasms (SI-NENs) is to avoid local recurrence while sparing as much of the small bowel as possible, even in the case of extensive mesenteric fibrosis. The results of PTR with retrograde vessel-sparing lymphadenectomy (VS-LA) were compared to those of conventional lymphadenectomy (Con-LA). METHODS: Prospectively collected clinical, surgical and pathological data of consecutive patients with SI-NENs who underwent small bowel resections were retrospectively analyzed regarding the resection technique performed. RESULTS: In a 7-year period, 50 of 102 patients with SI-NENs had only small bowel resections; of those, 25 were VS-LA and 25 were Con-LA. Patients with VS-LA had tendentially more advanced diseases with slightly higher rates of abdominal pain, mesenteric shrinkage and more level III lymph node involvement compared to patients with Con-LA. VS-LA, however, resulted in shorter resected bowel segments (median 40 cm vs. 65 cm, p = 0.007) with similar rates of local R0 resections (72% vs. 84%) and resected lymph nodes (median 13 vs. 13). Postoperative clinically relevant complications occurred in 1 of 25 (4%) in the VS-LA and in 7 of 25 (28%) patients in the Con-LA group (p = 0.02). Three months after surgery, 1 of 25 (4%) patients of the VS-LA group and 10 of 25 (40%) patients in the Con-LA group (p = 0.002) complained about abdominal pain. One of eight patients in the VS-LA group and two of thirteen patients in the Con-LA group who had completely resected stage III disease complained about diarrhea (p = 0.31). CONCLUSION: VS-LA seems to be oncologically safe and should be considered in small bowel resections for SI-NENs.

Heavier Weight of Resected Thyroid Specimen Is Associated With Higher Postoperative Morbidity in Benign Goiter.

OBJECTIVE: The impact of heavier weight of resected thyroid specimen on postoperative morbidity after total thyroidectomy for multinodular benign goiter remains unclear. METHODS: Data from the prospective StuDoQ|Thyroid registry of the German Society of General and Visceral Surgery were analyzed regarding the weight of the resected thyroid specimen and perioperative morbidity (vocal cord palsy, hemorrhage, surgical site infection, and hypocalcemia). To achieve a homogeneous patient population, only patients with total thyroidectomy for multinodular benign goiter were included. RESULTS: A total of 7911 patients from 105 departments underwent total thyroidectomy for benign conditions (January 2017-July 2020). The median resected weight of the thyroid specimen in all patients was 53 g (interquartile range 32-92). In 1732 patients, the specimen weight exceeded 100 g. Intraoperative neuromonitoring was used in 99.5% of patients. Postoperative laryngoscopy revealed vocal cord dysfunction in 480 of 15 822 (3.03%) nerves at risk, with unilateral dysfunction in 454 (2.87%) of patients and bilateral dysfunction in 13 patients (0.08%). In multivariable analysis, a thyroid weight >100 g was an independent predictor of early postoperative vocal cord dysfunction [odds ratio (OR) 1.462, 95% CI 1.108-1.930, P = 0.007). Heavier (>100 g) thyroid weight was an independent predictor of surgical site infection (OR 1.861, 95% CI 1.203-2.880, P = 0.005) and also predicted postoperative hemorrhage in the univariate analysis (OR 1.723, 95% CI 1.027-2.889, P = 0.039). On the contrary, postoperative parathyroid function was not affected. CONCLUSIONS: Heavier (>100 g) resected thyroid weight independently predicts higher postoperative morbidity, including early vocal cord palsy and surgical site infection after total thyroidectomy for benign multinodular goiter.

Regional Growth Velocity and Incidence of Pancreatic Neuroendocrine Neoplasias in Multiple Endocrine Neoplasia Type 1.

OBJECTIVES: Pancreatic neuroendocrine neoplasias (pNENs) in multiple endocrine neoplasia type 1 are predominantly found in the dorsal anlage. Whether their growth velocity and incidence might be related to their location in the pancreas has not been investigated yet. METHODS: We studied 117 patients using endoscopic ultrasound. RESULTS: Growth velocity could be calculated for 389 pNENs. Increase of largest tumor diameter (% per month) was 0.67 (standard deviation [SD], 2.04) in the pancreatic tail (n = 138), 1.12 (SD, 3.00) in the pancreatic body (n = 100), 0.58 (SD, 1.19) in the pancreatic head/uncinate process-dorsal anlage (n = 130), and 0.68 (SD, 0.77) in the pancreatic head/uncinate process-ventral anlage (n = 12). Comparing growth velocity of all pNENs in the dorsal (n = 368, 0.76 [SD, 2.13]) versus ventral anlage, no significant difference was detected. Annual tumor incidence rate was 0.21 in the pancreatic tail, 0.13 in the pancreatic body, 0.17 in the pancreatic head/uncinate process-dorsal anlage, 0.51 dorsal anlage together, and 0.02 in the pancreatic head/uncinate process-ventral anlage. CONCLUSIONS: Multiple endocrine neoplasia type 1 pNENs are unequally distributed between ventral (low prevalence and incidence) and dorsal anlage. However, there are no regional differences in growth behavior.

Long Non-Coding RNA H19 Expression Correlates with Autophagy Process in Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is characterized by poor prognosis and high mortality. The suppression of the long-non-coding RNA H19, counterbalanced by IGF2 over-expression, leads to down-regulation of the autophagy markers, high proliferation rate and metastatic potential in patients affected by ACC. The administration of the deacetylase inhibitors (DACi) panobinostat, trichostatin A (TSA) and SAHA affected the cell viability of H295R monolayer and spheroids and induced the over-expression of H19 and autophagy transcripts. H19 knock down in H295R cells was not able to modulate the expression level of autophagy transcripts. Instead, H19 knock down was able to impede the ability of DACi to modulate the protein level of the autophagy markers. Furthermore, the administration of higher concentration of DACi was able to down-regulate the protein level of Beclin1 and p62 and to induce the conversion of LC3B-I into the active LC3B-II form, thus confirming an active autophagic process. Neither the active protein level nor the activity of caspases 8 and 3 was prompted by the DACi, thus excluding the involvement of the executioners of apoptosis in H295R decay. The DACi restore H19, the autophagy markers and trigger cell death in ACC cells. The re-activation of autophagy would represent a novel strategy for the treatment of patients affected by this severe malignancy.

Relevant prognostic factors in patients with stage IV small intestine neuroendocrine neoplasms.

There are few, but controversial data on the prognostic role of upfront primary tumour resection and mesenteric lymph node dissection (PTR) in patients with diffuse metastatic small intestinal neuroendocrine neoplasia (SI-NEN). Therefore, the prognostic role of PTR and other factors was determined in this setting. This retrospective cohort study included patients with stage IV SI-NETs with unresectable distant metastases without clinical and radiological signs of acute bowel obstruction or ischaemia. Patients diagnosed from January 2002 to May 2020 were retrieved from a prospective SI-NEN database. Disease specific overall survival (OS) was analysed with regard to upfront PTR and a variety of other clinical (e.g., gender, age, Hedinger disease, carcinoid syndrome, diarrhoea, laboratory parameters, metastatic liver burden, extrahepatic and extra-abdominal metastasis) and pathological (e.g., grading, mesenteric gathering) parameters by uni- and multivariate analysis. A total of 138 patients (60 females, 43.5%) with a median age of 60 years, of whom 101 (73%) underwent PTR and 37 (27%) did not, were included in the analysis. Median OS was 106 (95% CI: 72.52-139.48) months in the PTR group and 52 (95% CI: 30.55-73.46) in the non-PTR group (p = 0.024), but the non-PTR group had more advanced metastatic disease (metastatic liver burden ≥50% 32.4% vs. 13.9%). There was no significant difference between groups regarding the rate of surgery for bowel complications during a median follow-up of 51 months (PTR group 10.9% and non-PTR group 16.2%, p = 0.403). Multivariate analysis revealed age < 60 years, normal C-reactive protein (CRP) at baseline, absence of diarrhoea, less than 50% of metastatic liver burden, and treatment with PRRT as independent positive prognostic factors, whereas PTR showed a strong tendency towards better OS, but level of significance was missed (p = 0.067). However, patients who underwent both, PTR and peptide radioreceptor therapy (PRRT) had the best survival compared to the rest (137 vs. 73 months, p = 0.013). PTR in combination with PRRT significantly prolongs survival in patients with stage IV SI-NEN. Prophylactic PTR does also not result in a lower reoperation rate compared to the non-PTR approach regarding bowel complications.

Pancreatic Neuroendocrine Neoplasias in Multiple Endocrine Neoplasia Type 1 Are Predominantly Located in the Dorsal Anlage: An Endoscopic Ultrasound Study.

OBJECTIVES: Pancreatic neuroendocrine neoplasias (pNENs) frequently occur in multiple endocrine neoplasia type 1 (MEN1). Their distribution referring to embryology, that is, the pancreatic anlagen, has not been investigated yet. METHODS: In the time between 1998 and 2019, we studied the distribution of pNENs in MEN1 concerning the embryologic origin of the pancreas, that is, the dorsal versus ventral anlage using endoscopic ultrasound in 117 MEN1 patients: 56 women, 61 men; aged 40 years (standard deviation, 14 years) at first endoscopic ultrasound. RESULTS: In 105 patients, a total of 628 pNENs were detected. They were located in the pancreatic tail: 231; pancreatic body: 177; pancreatic head/uncinate process: 220. Of the latter, 22 were located in the ventral anlage, 176 in the dorsal anlage, and 22 remained undefined. In summary, just 3.5% of all detected pNENs were located in the ventral anlage, 93.0% in the dorsal anlage, and 3.5% could not be assigned. CONCLUSIONS: Our study indicates that the vast majority of pNENs in MEN1 is located in the dorsal anlage, whereas the ventral anlage of the pancreas seems to be to a large extend spared from pNENs. Implications for new surgical strategies might be considered.

Short-Term Hypocalcemia Prophylaxis With Calcitriol Before Thyroidectomy.

BACKGROUND: Total thyroidectomy is the most common surgical treatment of thyroid diseases, and postoperative hypocalcemia is its most common complication. Hypocalcemia prolongs the patient's hospital stay and impairs his or her quality of life. Although a low vitamin D level is a recognized risk factor, the utility of preoperative vitamin D administration to prevent postoperative hypocalcemia is unclear. In this trial, therefore, we studied the effect of giving vitamin D before total thyroidectomy. METHODS: In a multicenter, randomized, minimally interventional trial (registration number: DRKS 00005615), patients about to undergo total thyroidectomy were randomized either to an intervention group that received 0.5 µg of calcitriol per os twice daily for three days up to the day immediately before surgery, or to a control group that did not (no placebo was given). The primary endpoint was the absence of hypocalcemia (serum calcium <2.1 mmol/L) in the postoperative course. RESULTS: Of the 287 patients recruited in six hospitals over the period 23 July 2014 to 20 March 2017, 246 were included in the final analysis. The intervention and control groups did not differ significantly with respect to the rate of postoperative hypocalcemia (29.2% and 33.6%, respectively; p = 0.546, power 8.8%). The duration of postoperative hypocalcemia was, however, shorter in the intervention group (3.5 vs. 7 days; p = 0.016, power 68%). The rates of hypocalcemia in the individual trial locations varied widely, ranging from 13.9% to 71.4%. CONCLUSION: Short-term administration of calcitriol did not affect the rate of occurrence of hypocalcemia after thyroidectomy, but did shorten its duration. The rate of postoperative hypocalcemia varied widely across hospitals, probably because of differences in surgical technique.

Tracking the Distribution of Brucella abortus in Egypt Based on Core Genome SNP Analysis and In Silico MLVA-16.

Brucellosis, caused by the bacteria of the genus Brucella, is one of the most neglected common zoonotic diseases globally with a public health significance and a high economic loss among the livestock industry worldwide. Since little is known about the distribution of B. abortus in Egypt, a total of 46 B. abortus isolates recovered between 2012-2020, plus one animal isolate from 2006, were analyzed by examining the whole core genome single nucleotide polymorphism (cgSNP) in comparison to the in silico multilocus variable number of tandem repeat analysis (MLVA). Both cgSNP analysis and MLVA revealed three clusters and one isolate only was distantly related to the others. One cluster identified a rather widely distributed outbreak strain which is repeatedly occurring for at least 16 years with marginal deviations in cgSNP analysis. The other cluster of isolates represents a rather newly introduced outbreak strain. A separate cluster comprised RB51 vaccine related strains, isolated from aborted material. The comparison with MLVA data sets from public databases reveals one near relative from Argentina to the oldest outbreak strain and a related strain from Spain to a newly introduced outbreak strain in Egypt. The distantly related isolate matches with a strain from Portugal in the MLVA profile. Based on cgSNP analysis the oldest outbreak strain clusters with strains from the UK. Compared to the in silico analysis of MLVA, cgSNP analysis using WGS data provides a much higher resolution of genotypes and, when correlated to the associated epidemiological metadata, cgSNP analysis allows the differentiation of outbreaks by defining different outbreak strains. In this respect, MLVA data are error-prone and can lead to incorrect interpretations of outbreak events.

Carbonic-anhydrase IX expression is increased in thyroid cancer tissue and represents a potential therapeutic target to eradicate thyroid tumor-initiating cells.

The expression of Carbonic-anhydrase IX (CAIX) in thyroid cancer (TC) subtypes was determined and its role in cancer cell growth and tumor-initiating cells (TICs) investigated. Immunohistochemistry in 114 TC patients revealed that CAIX expression was increased in tumor specimens of papillary, follicular and anaplastic TCs compared to normal thyroid tissue. Clinicopathological data indicated that lymph node metastases were more frequent in patients with high CAIX expression. The Cancer Genome Atlas database analysis demonstrated that a strong CAIX-mRNA expression was associated with advanced tumor stages and poor survival in TCs. In TC cell lines, CAIX expression was elevated in tumorspheres compared to monolayer cultures and was associated with an increased expression of stemness markers. Genetic knockdown or pharmacological inhibition of CAIX suppressed cell proliferation and the TIC ability to form tumorspheres by an induction of apoptosis and cell-cycle arrest. These findings suggest CAIX as a potential prognostic marker and a therapeutic target for thyroid cancer.

Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53.

Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein-protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.

Molecular characterization and antimicrobial susceptibility testing of clinical and non-clinical Brucella melitensis and Brucella abortus isolates from Egypt.

Brucellosis is a highly contagious and incapacitating disease of humans, livestock and wildlife species globally. Treatment of brucellosis in animals is not recommended, and in humans, combinations of antibiotics recommended by the World Health Organization are used. However, sporadic antimicrobial-resistant (AMR) isolates and relapse cases have been reported from different endemic regions. In the current study, molecular characterization and antibiotic susceptibility testing using the microdilution method for 35 B. abortus and B. melitensis strains isolated from humans, milk and animal were carried out. Additionally, Next-Generation-Sequencing (NGS) technology was applied to confirm Brucella at the species level and investigate AMR and pathogenicity-associated determinants. MALDI-TOF seemed to be a rapid and reliable tool for routine identification of brucellae to the genus level; however, DNA-based identification is indispensable for accurate species identification. Brucella abortus strains were isolated from two human cases and a sheep. Such infections are uncommon in Egypt. Egyptian Brucella strains are still in-vitro susceptible to doxycycline, tetracyclines, gentamicin, ciprofloxacin, levofloxacin, chloramphenicol, streptomycin, trimethoprim/sulfamethoxazole and tigecycline. Probable (no CLSI/EUCAST breakpoints have been defined yet) in-vitro resistance to rifampicin and azithromycin was observed. WGS failed to determine classical AMR genes, and no difference in the distribution of virulence-associated genes in all isolates was found. Isolates of human and non-human origins were still susceptible to the majority of antibiotics used for treatment in humans. The absence of classical AMR genes in genomes of "resistant" Brucella strains may reflect a lack of information in databases, or resistance might not be encoded by single resistance genes. The One Health approach is necessary for tackling brucellosis. Continuous susceptibility testing, updating of breakpoints, assessing mutations that lead to resistance are needed.

Long-term outcome of surgical resection in patients with gastroenteropancreatic neuroendocrine neoplasia: results from a German nation-wide multi-centric registry.

BACKGROUND: Neuroendocrine neoplasia (NEN) are rare and heterogenous tumours. Few data exist on the impact of surgical therapy. MATERIALS AND METHODS: This is a retrospective analysis of prospectively collected data of gastroenteropancreatic NEN in the German NET-Registry (1999-2012). It focuses on patients without distant metastases (limited disease, LD, stage I-IIIB). RESULTS: Data of 2239 patients with NEN were recorded. Median age was 59 years, the gender ratio was 1:1.3 (f:m). A total of 986 patients (44%) had LD, and the 5-year survival rate (5 years) was 77% for all and 90% for patients with LD. A total of 1635 patients (73%) received a surgical therapy (1st to 6th line); the 5 and 10 ysr were 83/65% after and 59/35% without surgery for all patients (p < .001). The resection margins in the LD patients were 76%, 16%, and 3% for R0, R1 and R2, respectively. The 10 ysr was 84%, 59% and 42% for R0, R1 and R2 resections, respectively (p = .021 R0/R1, p < .001 R0/R2). The R0 resection rate was 75% for G1/G2 NET and 67% for G3 NEC. CONCLUSION: The rate of complete tumour resection (R0) in LD is independent of tumour grading, and R0 resection is the key determinant of long-term survival, as demonstrated by the 10 ysr. of 84%. All NEN patients with limited disease should be considered for operation, if possible, as the best 10-year survival is shown after an R0 resection.