Bacterial strains of the genus Geobacillus grow at high temperatures of 50-75 °C and could thus be useful for biotechnological applications. However, genetic manipulation of these species is ...difficult because the current techniques for transforming Geobacillus species are not efficient. In this study, we developed an easy and efficient method for transforming Geobacillus kaustophilus using the conjugative plasmid pLS20cat.
We constructed a transformation system comprising (i) a mobilizable Bacillus subtilis-G. kaustophilus shuttle plasmid named pGK1 that carries the elements for selection and replication in Geobacillus, and (ii) a pLS20cat-harboring B. subtilis donor strain expressing the dam methylase gene of Escherichia coli and the conjugation-stimulating rap
gene of pLS20cat. This system can be used to efficiently introduce pGK1 into G. kaustophilus by mobilization in a pLS20cat-dependent way. Whereas the thermostable kanamycin marker and Geobacillus replication origin of pGK1 as well as expression of dam methylase in the donor were indispensable for mobilization, ectopic expression of rap
increased its efficiency. In addition, the conditions of the recipient influenced mobilization efficiency: the highest mobilization efficiencies were obtained using recipient cells that were in the exponential growth phase. Furthermore, elimination of the origin of transfer from pLS20cat enhanced the mobilization.
We describe a novel method of plasmid mobilization into G. kaustophilus recipient from B. subtilis donor depending on the helper function of pLS20cat, which enables simple, rapid, and easy transformation of the thermophilic Gram-positive bacterium.
The conjugative plasmid, pLS20, isolated from Bacillus subtilis natto, has an outstanding capacity for rapid self-transfer. In addition, it can function as a helper plasmid, mediating the ...mobilization of an independently replicating co-resident plasmid.
In this study, the oriT sequence of pLS20cat (oriT
) was eliminated to obtain the plasmid, pLS20catΔoriT. This resulted in the complete loss of the conjugative transfer of the plasmid but still allowed it to mobilize a co-resident mobilizable plasmid. Moreover, pLS20catΔoriT was able to mobilize longer DNA segments, up to 113 kb of chromosomal DNA containing oriT
, after mixing the liquid cultures of the donor and recipient for only 15 min.
The chromosomal DNA mobilization mediated by pLS20catΔoriT will allow us to develop a novel genetic tool for the rapid, easy, and repetitive mobilization of longer DNA segments into a recipient chromosome.
Intratympanic steroid injection (ITSI) can be an effective treatment for sudden sensorineural hearing loss or Meniere's disease. Tympanic membrane (TM) perforation after ITSI is a major complication ...which needs additional treatment. The purpose of this study is to assess the factors associated with TM perforation after ITSI.
We obtained the clinical data of patients who underwent ITSI treatment at the Department of Otolaryngology JR Tokyo General Hospital from April 2013 to March 2021. The data included age, sex, treated side, number of injections, average interval between injections, TM anesthesia with Zentöl solution, which contains phenol, any history of diabetes and any concurrent use of oral or intravenous steroids. We evaluated the association between these variables and TM perforation after ITSI using the Student's t-test, the chi-squared test, univariate logistic regression analysis and multivariate logistic regression analysis. TM perforation was defined as a case in which perforation was observed at least once during outpatient visits.
Records of 205 ears in 190 patients were analyzed. The overall proportion of TM perforation in the early period after ITSI was 12.7% (26 out of 205 ears), which decreased to 9.3% (19 out of 205 ears) and 5.9% (12 out of 205 ears) at the 1- and 3-month follow-ups, respectively. The proportion of TM perforation in the early period after ITSI without TM anesthesia was 3.5% (5 ears out of 145 ears), which decreased to 1.4% (2 ears) or 0% at the 1- or 3- month follow-ups, respectively. The use of tympanic anesthetics which contain phenol was significantly associated with TM perforation in univariate logistic regression analysis (odds ratio: 15.08, 95% confidence interval: 5.34–42.56, p < 0.001) and in multivariate analysis (odds ratio: 20.76, 95% confidence interval: 6.31–68.3, p < 0.001). All TM perforation cases without TM anesthesia healed spontaneously or with paper tympanic closure treatment. TM perforation in 6 ears out of 21 ears with TM anesthesia did not heal during the follow-up.
The overall proportion of TM perforations from the early period after ITSI was 12.7%, 9.3% at the 1-month post-ITSI outpatient follow-up, 5.9% at the 3-month post-ITSI outpatient follow-up. Tympanic anesthesia was significantly associated with TM perforation after ITSI, which indicated that TM anesthesia with solutions containing phenol is not recommended for ITSI.
Cholangiocarcinoma (CCA) is an aggressive tumor with limited treatment options especially in 2nd line or later treatments. Targeting fibroblast growth factor receptor (FGFR) 2 has recently emerged as ...a promising treatment option for patients with CCA harboring FGFR2-fusion. This study investigated the antitumor activities of tasurgratinib as an orally available FGFR1-3 inhibitor, in preclinical FGFR2-driven CCA models.
Antitumor activities of tasurgratinib were examined in vitro and in vivo using NIH/3T3 cells expressing FGFR2-fusion as FGFR2-driven CCA models, and in vivo using a CCA patient-derived xenograft model. The molecular mechanism of action of tasurgratinib was elucidated through co-crystal structure analysis with FGFR1, manual complex model analysis with FGFR2, and binding kinetics analysis with FGFR2. Furthermore, the cell-based inhibitory activities against acquired resistant FGFR2 mutations in patients with CCA treated with FGFR inhibitors were evaluated.
Tasurgratinib showed antitumor activity in preclinical FGFR2-driven CCA models by inhibiting the FGFR signaling pathway in vitro and in vivo. Furthermore, cell-based target engagement assays indicated that tasurgratinib had potent inhibitory activities against FGFR2 mutations, such as N549H/K, which are the major acquired mutations in CCA. We also confirmed that tasurgratinib exhibited fast association and slow dissociation kinetics with FGFR2, binding to the ATP-binding site and the neighboring region, and adopting an Asp-Phe-Gly (DFG)-"in" conformation.
These data demonstrate the therapeutic potential of tasurgratinib in FGFR2-driven CCA and provide molecular mechanistic insights into its unique inhibitory profile against secondary FGFR2 resistance mutations in patients with CCA treated with FGFR inhibitors.
Inherited bone-marrow-failure syndromes (IBMFSs) include heterogeneous genetic disorders characterized by bone-marrow failure, congenital anomalies, and an increased risk of malignancy. Many lines of ...evidence have suggested that p53 activation might be central to the pathogenesis of IBMFSs, including Diamond-Blackfan anemia (DBA) and dyskeratosis congenita (DC). However, the exact role of p53 activation in each clinical feature remains unknown. Here, we report unique de novo TP53 germline variants found in two individuals with an IBMFS accompanied by hypogammaglobulinemia, growth retardation, and microcephaly mimicking DBA and DC. TP53 is a tumor-suppressor gene most frequently mutated in human cancers, and occasional germline variants occur in Li-Fraumeni cancer-predisposition syndrome. Most of these mutations affect the core DNA-binding domain, leading to compromised transcriptional activities. In contrast, the variants found in the two individuals studied here caused the same truncation of the protein, resulting in the loss of 32 residues from the C-terminal domain (CTD). Unexpectedly, the p53 mutant had augmented transcriptional activities, an observation not previously described in humans. When we expressed this mutant in zebrafish and human-induced pluripotent stem cells, we observed impaired erythrocyte production. These findings together with close similarities to published knock-in mouse models of TP53 lacking the CTD demonstrate that the CTD-truncation mutations of TP53 cause IBMFS, providing important insights into the previously postulated connection between p53 and IBMFSs.
We have previously reported that the low rate of osteoporosis patients treated with anti-osteoporotic drugs following surgical treatment for the first fragility fractures by orthopaedic surgeons ...during 3 years from 2000 to 2003 was only 13.1%. Ten years have now passed our previous study, and we hypothesized that the rate of appropriate pharmacologic treatment for the prevention of secondary fractures has improved.
We studied 730 osteoporosis patients (102 men and 628 women; average age of 78 years, range 33-102 years) who underwent surgical treatment for fragility fractures, during 3-year period from 2010 to 2012. The 730 cases consisted of 489 hip fractures and 241 distal radius fractures. All patients were admitted and underwent surgical intervention in hospitals. Variables were examined to ascertain whether pharmaceutical treatment was performed after discharge. Based on these data, we compared results for patients in the present study with those from our previous study.
The rate of treatment with anti-osteoporosis medication in the present (16.2%) was slightly but significantly improved from that in our previous study (13.1%). The rate of pharmaceutical treatment following hip fractures increased significantly, while that following distal radius fractures showed no significant change. Regarding the categories of anti-osteoporotic drugs prescribed to the patients, the rate of treatment with bisphosphonate as a higher evidenced drug for the prevention of fractures in the present study was significantly higher than that in our previous study.
We demonstrated that the rate of pharmacologic treatment by orthopaedic surgeons and the rate of more effective anti-osteoporotic drugs prescribed to the patients following surgical intervention for the first fragility fracture in the present study were improved in comparison with those of 10 years ago.
We report two cases of pyogenic spondylodiscitis caused by bacteremia following gastric surgery. Case 1 An 85-year-old male patient underwent total gastrectomy for gastric cancer. After the surgery, ...leukocytosis and elevated C reactive protein (CRP) were sustained; however, there was no surgical site infection (SSI). His lumbar pain was present; therefore, we performed magnetic resonance imaging (MRI). Thereafter, he was diagnosed with L3 spondylitis, L2/3 discitis, and bilateral iliopsoas abscess on postoperative day (POD) 33. He has been treated with daptomycin (DAP). Case 2 A 72-year-old male patient was admitted to our department for post-distal gastrectomy reflux esophagitis. After admission, conservative therapy was continued; however, severe symptoms appeared many times. Then, surgical treatment was scheduled to reduce reflux. When his body temperature (BT) was elevated to 39.1°C before the surgery, vancomycin (VCM) was administered because of suspected catheter infection. Once his BT normalized, fundoplication was performed. On POD 19, his BT elevated again, and the central vein (CV) catheter was removed. On POD 27, he complained of back pain. He was diagnosed with pyogenic spodylodiscitis using MRI. He was treated conservatively with VCM followed by sulbactam/ampicillin (SBT/ABP) based on the result of the culture.Physicians should be alert regarding possible occurrence of pyogenic spondylitis in patients with back pain or lumbago who have undergone gastric surgery.
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