Experimental Evolution of Copper Resistance in Escherichia coli Produces Evolutionary Trade-Offs in the Antibiotics Chloramphenicol, Bacitracin, and Sulfonamide.

The rise in antimicrobial resistant bacteria have prompted the need for antibiotic alternatives. To address this problem, significant attention has been given to the antimicrobial use and novel applications of copper. As novel applications of antimicrobial copper increase, it is important to investigate how bacteria may adapt to copper over time. Here, we used experimental evolution with re-sequencing (EER-seq) and RNA-sequencing to study the evolution of copper resistance in Escherichia coli. Subsequently, we tested whether copper resistance led to rifampicin, chloramphenicol, bacitracin, and/or sulfonamide resistance. Our results demonstrate that E. coli is capable of rapidly evolving resistance to CuSO4 after 37 days of selection. We also identified multiple de novo mutations and differential gene expression patterns associated with copper, most notably those mutations identified in the cpx gene. Furthermore, we found that the copper resistant bacteria had decreased sensitivity when compared to the ancestors in the presence of chloramphenicol, bacitracin, and sulfonamide. Our data suggest that the selection of copper resistance may inhibit growth in the antimicrobials tested, resulting in evolutionary trade-offs. The results of our study may have important implications as we consider the antimicrobial use of copper and how bacteria may respond to increased use over time.

Securing the surgical field for mobilization of right-sided colon cancer using the duodenum-first multidirectional approach in laparoscopic surgery.

BACKGROUND: The aim of this study was to compare the short-term outcomes of the duodenum-first multidirectional approach (DMA) in laparoscopic right colectomy with those of the conventional medial approach to assess its safety and feasibility. METHODS: This retrospective study enrolled 120 patients who had laparoscopic surgery for right-sided colon cancer in our institution between April 2013 and December 2019. Fifty-four patients underwent colectomy using the multidirectional approach; among these, 20 underwent the DMA and 34 underwent the caudal-first multidirectional approach (CMA). Sixty-six patients underwent the conventional medial approach. Complications within 30 days of surgery were compared between the groups. RESULTS: There were 54 patients in the multidirectional group [29 females, median age 72 years (range 36-91 years)] and 66 in the medial group [42 females, median age 72 years (range 41-91 years)]. Total operative time was significantly shorter in multidirectional approach patients than conventional medial approach patients (208 min vs. 271 min; p = 0.01) and significantly shorter in patients who underwent the DMA compared to the CMA (201 min vs. 269 min; p < 0.001). Operative time for the mobilization procedure was also significantly shorter in patients who underwent the DMA (131 min vs. 181 min; p < 0.001). Blood loss and incidence of postoperative complications did not differ. In 77 patients with advanced T3/T4 tumors, the DMA, CMA, and conventional medial approach were performed in 13, 21, and 43 patients, respectively. Total operative time and operative time of the mobilization procedure were significantly shorter in patients undergoing DMA. Blood loss and incidence of postoperative complications did not differ. R0 resection was achieved in all patients with advanced tumors. CONCLUSIONS: The DMA in laparoscopic right colectomy is safe and feasible and can achieve R0 resection with a shorter operative time than the conventional medial approach, even in patients with advanced tumors.

Accelerated food source location in aging Drosophila.

Adequate energy stores are essential for survival, and sophisticated neuroendocrine mechanisms evolved to stimulate foraging in response to nutrient deprivation. Food search behavior is usually investigated in young animals, and it is not known how aging alters this behavior. To address this question in Drosophila melanogaster, we compared the ability to locate food by olfaction in young and old flies using a food-filled trap. As aging is associated with a decline in motor functions, learning, and memory, we expected that aged flies would take longer to enter the food trap than their young counterparts. Surprisingly, old flies located food with significantly shorter latency than young ones. Robust food search behavior was associated with significantly lower fat reserves and lower starvation resistance in old flies. Food-finding latency (FFL) was shortened in young wild-type flies that were starved until their fat was depleted but also in heterozygous chico mutants with reduced insulin receptor activity and higher fat deposits. Conversely, food trap entry was delayed in old flies with increased insulin signaling. Our results suggest that the difference in FFL between young and old flies is linked to age-dependent differences in metabolic status and may be mediated by reduced insulin signaling.

SLA-DRB1 and -DQB1 genotyping by the PCR-SSOP-Luminex method.

A simple and novel genotyping method was developed to detect alleles at the swine leukocyte antigen (SLA)-DRB1 and -DQB1 class II loci by using polymerase chain reaction (PCR)-fluorescently labeled sequence-specific oligonucleotide probes (SSOPs) and Luminex 100 xMAP detection. The PCR-SSOP-Luminex method exhibited accuracy of 95% for both SLA-DRB1 and -DQB1 in 6 homozygous and 16 heterozygous pig samples as confirmed by sequencing the PCR products of the same samples. In addition, 12 low-resolution SLA class II haplotypes consisting of 7 and 9 DRB1 and DQB1 alleles were identified, respectively, in one population of 283 Landrace pigs. This genotyping method facilitates the rapid and accurate identification of two- or four-digit alleles at the SLA-DRB1 and -DQB1 loci.

GM1/GalNAc-GD1a complex: a target for pure motor Guillain-Barre syndrome.

BACKGROUND: GM1 and GalNAc-GD1a are located on the axolemma of the motor nerves and are believed to be the antigens associated with pure motor Guillain-Barré syndrome (GBS). Furthermore, GM1 and GalNAc-GD1a may exist nearby and colocalize on the axolemma. Ganglioside complex (GSC) antigens associated with GM1 or GalNAc-GD1a can be target antigens in pure motor GBS. We investigated GBS sera for antibodies to a GSC consisting of GM1 and GalNAc-GD1a (GM1/GalNAc-GD1a) and analyzed the clinical and electrophysiologic findings of patients with antibodies to GM1/GalNAc-GD1a. METHODS: Sera from 224 patients with GBS were surveyed for antibodies to GSCs consisting of two of nine gangliosides (GM1, GM2, GM3, GD1a, GD3, GT1a, GT1b, GQ1b, and GalNAc-GD1a). We analyzed the clinical and electrophysiologic features of patients with IgG antibodies to the GM1/GalNAc-GD1a complex. RESULTS: Ten patients with GBS had IgG antibodies to the GM1/GalNAc-GD1a complex. The clinical findings of the 10 patients with GBS were characterized by preserved sensory system and infrequent cranial nerve deficits. According to the criteria established by Hadden et al., electrodiagnostic studies showed a demyelinating pattern in four patients and axonal neuropathy pattern in two. Early motor conduction block at intermediate nerve segments was found in five patients. CONCLUSIONS: GM1 and GalNAc-GD1a may form a complex in the axolemma at nodes of Ranvier or paranodes of the motor nerves, and may be a target antigen in pure motor Guillain-Barré syndrome, especially in the form of acute motor conduction block neuropathy.

Intra-bone marrow cotransplantation of donor mesenchymal stem cells in pig-to-NOD/SCID mouse bone marrow transplantation facilitates short-term xenogeneic hematopoietic engraftment.

We directly injected porcine donor mesenchymal stem cells (MSC) into murine bone marrow (BM) cavities to examine the effects of intra-BM cotransplantation of MSC in pig-to-NOD/SCID mouse bone marrow transplantation (BMT) on xenogeneic engraftment. Porcine MSC prepared by aspiration of iliac BM of miniature swine were identified as CD90+CD29+CD45-CD31- and shown to differentiate into osteoblastocytes and adipocytes. A few weeks after expansion, MSC (1 x 10(6) cells/mouse) were directly injected with BM cells (30 x 10(6) cells/mouse) obtained from vertebrae through a microsyringe into BM cavities of both tibiae of NOD/SCID mice after 3-Gy total body irradiation. Controls were injected with only BM cells. Porcine chimerisms of BM cells of tibiae (injection site) and of femurs (non-injection site) in recipient mice were evaluated with porcine and murine cell markers using FACS. The chimerism of porcine class I+ cells at the injection site in the MSC group and the controls were 3.45%, 1.43%, and 0.17%, and 2.27%, 0.81%, and 0.1% at 1, 3, and 6 weeks, respectively. The chimerism at the noninjection site in the MSC group and the controls were 0.21%, 1.34%, and 0.11%, and 0.06%, 0.42%, and 0.09% at 1, 3, and 6 weeks, respectively. The total chimerisms of injection site in the MSC group to 6 weeks were significantly higher than those in the control group (1.60% vs 0.99%; P < .05), whereas the chimerism of the noninjection site in MSC group was remarkably higher at 3 weeks. In conclusion, intra-BM cotransplantation of porcine donor MSC in pig-to-NOD/SCID mouse BMT improved short-term xenogeneic engraftment, presumably due to humoral factors.

Genetic analysis on HLA loci in Japanese patients with abdominal aortic aneurysm.

PURPOSE: autoimmunity has been proposed as one of the pathogenesis of abdominal aortic aneurysm (AAA). There is also a likelihood that when aorto-iliac occlusive disease (AIOD) coexists with AAA, some other occlusive atherosclerotic diseases, such as ischemic heart disease and cerebrovascular disease, may develop, leading to a very poor long-term prognosis. Previous studies using serological HLA typing showed that HLA-DR15 was a risk factor for AAA. In this study, we performed HLA-DNA typing by PCR to clarify the relationship between AAA and HLA genotypes in Japanese patients with AAA. In addition, we analyzed whether HLA genotypes are involved in the pathogenesis of AIOD. RESULTS: we examined 78 HLA genotypes of class I (HLA-A and -B) and class II (HLA-DR) and found that 60.4 and 30.4% of 49 AAA patients had HLA-A2 and HLA-B61, respectively. These frequencies were significantly higher than those in control individuals (HLA-A2, p < 0.05; HLA-B61, p < 0.005). We also found that 55.6% of nine AAA patients with AIOD had both HLA-B52 and HLA-DR B1*1502. In contrast, only 10.0% each of 40 AAA patients without AIOD showed HLA-B53 or HLA-DR B1*1502. CONCLUSIONS: this study showed that HLA A-2 and HLA B-61, but not HLA DR-15, were important genetic risk factors for the development of AAA among the Japanese population. We also found high frequencies of HLA-B52 and HLA-DR B1*1502 in the AAA patients with AIOD than in those without, although this must be confirmed using a larger number of AAA patients with AIOD.

Susceptibility locus for non-obstructive azoospermia is localized within the HLA-DR/DQ subregion: primary role of DQB1*0604.

Non-obstructive azoospermia is a male infertility characterized by no or little sperm in semen as a result of a congenital dysfunction in spermatogenesis. Previous studies have reported a higher prevalence of particular human leukocyte antigen (HLA) antigens in non-obstructive azoospermia. As the expression of the RING3 gene located in the HLA class II region was predominant in the testis, mainly around spermatids and pachytene spermatocytes, it is tempting to speculate that RING3 is one of the strong candidate genes responsible for the pathogenesis of the disease. In this study, the genetic polymorphism in the RING3 gene was investigated by the direct sequencing technique. As a result, a total of 14 single nucleotide polymorphisms were identified. Among them, six were localized in the coding region but none of them was accompanied by an amino-acid substitution. No significant difference in the allelic distribution at these 14 polymorphic sites was observed between the patients and healthy controls, suggesting that the susceptible gene for non-obstructive azoospermia is not the RING3 gene. Then, in order to map the susceptibility locus for non-obstructive azoospermia precisely within the HLA region, 11 polymorphic microsatellite markers distributed from the SACM2L gene just outside the HLA class II region (187 kb telomeric of the DPB1 gene) to the OTF3 gene in the HLA class I region were subjected to association analysis in the patients. Statistical analysis of distribution in the allelic frequency at each microsatellite locus demonstrated that the pathogenic gene for non-obstructive azoospermia is located within the HLA-DR/DQ subregion. In fact, DRB1*1302 and DQB1*0604 were found to be strongly associated with non-obstructive azoospermia by polymerase chain reaction-based DNA typing. Further, haplotype analysis suggested that the DQB1*0604 allele may play a decisive role in the pathogenesis of non-obstructive azoospermia.

Susceptibility to vesicoureteral reflux in Japanese is linked to HLA-DR antigen.

OBJECTIVES: To determine whether vesicoureteral reflux is associated with the human leukocyte antigen (HLA) genes. METHODS: We evaluated 40 Japanese patients (27 males and 13 females) with reflux. HLA-DR low-resolution genotyping and high-resolution typing of HLA-DRB1 alleles were performed. The frequencies of the HLAs and alleles were calculated and compared with those previously reported in 493 healthy Japanese. RESULTS: Low-resolution typing showed that the frequency of the HLA-DR11 antigen was significantly higher in the patients with reflux than in the control group. High-resolution typing revealed that the frequencies of HLA-DRB1*1101 and 1502 alleles were significantly higher in the patients with reflux than in the control group. In the patients with and without renal scarring, the frequencies of the HLA-DR11 antigens and HLA-DRB1*1101 alleles were significantly lower in those with renal scarring. In the patients with and without the chief complaint of urinary tract infection symptoms, the frequencies of HLA-DR13 antigens and HLA-DRB1*1302 alleles were significantly lower in those with that chief complaint. CONCLUSIONS: The susceptibility to reflux is, in part, controlled by HLA genes themselves or an unknown gene or genes, the locus for which is located close to the DRB1 gene. The lack of a HLA-DRB1*1101 allele and DRB1*1302 allele in patients with reflux might be connected with renal scarring and urinary tract infection, respectively.

cDNA cloning and genetic polymorphism of the swine major histocompatibility complex (SLA) class II DMA gene.

cDNA clones corresponding to the swine histocompatibility complex (SLA: swine leucocyte antigen)-DM alpha chain were isolated using the polymerase chain reaction (PCR) products from the third exon in the human HLA-DMA gene as a probe. Amino acid comparative analysis revealed that these clones were more closely related to the bovine and human DMA genes than to the other swine class II genes alpha chain genes, DRA, DQA and DOA. These results suggest that the SLA-DMA gene is expressed and may function, like HLA-DM, as an important modulator in class II restricted antigen processing in swine. Furthermore, based on the sequences and PCR-restriction fragment length polymorphism (PCR-RFLP) patterns in the SLA-DMA gene, no allelic variation was recognized in the second exon, but five allelic variations were recognized in the third exon in five different breeds of swine. These DMA alleles were defined by variation at four nucleotide positions. Two of these alleles resulted in an amino acid substitution. These results suggest that SLA-DMA has little polymorphism as observed in HLA-DMA and mouse H2-Ma.

On the MICA deleted-MICB null, HLA-B*4801 haplotype.

A 100-kb deletion including the MICA gene was recently reported in the HLA-B48 (B*4801)-associated haplotype in Japanese. Interestingly, this MICA deletion is accompanied by a MICB null allele, MICB0107N. In order to further investigate the universality of the apparent tight linkage between these two events, we present data on high-resolution deletion mapping of eight HLA-B48-homozygous individuals. Among these, five carried the MICA deletion linked to MICB0107N, as originally reported. Conversely, the remaining three possessed an intact MICA gene of MICA008 or MICA010 allelic variant associated this time with a putative expressed MICB allele, MICB0102. These results may imply that the expression of both MICA and MICB molecules is indispensable to viability through a yet-to-be understood mutual interaction in immune surveillance.

[Cystic angiomatosis]. / Angiomatosis quística.

A case of cystic angiomatosis is presented in a patient whose clinical sign were the presence of an inguinoescrotal mass due to a retroperitoneal lymphangioma, visceral splenic lesions and diffuse skeletal lesions. This rare entity is discussed and a brief review of the literature is made.

HLA-DRB genotypes in Japanese patients with renal cell carcinoma.

OBJECTIVES: Several clinical features, such as the spontaneous regression of some renal cell carcinoma (RCC) metastases after nephrectomy, suggest immune involvement in tumor destruction. PATIENTS AND METHODS: We investigated the role of genetic variation at the HLA class II loci in RCC by analyzing the HLA-DR antigen and HLA-DRB1, DRB3, DRB4 and DRB5 alleles in 55 patients using the polymerase chain rection. RESULTS: No statistically significant differences were observed in the frequency of the HLA-DR antigen or HLA-DRB3, DRB4, or DRB5 genes between the patients and a healthy control group. On the other hand, the HLA-DRB1*0403 and *1202 alleles were significantly more frequent in the patients than in the controls (p < 0.001 and p < 0.01, respectively). CONCLUSION: RCC might be linked to these two alleles in Japanese patients.

Fracture of hamate hook--diagnosis by the hamate hook lateral view.

Ten intact wrist joints were examined to evaluate the hamate hook visualisation by a hamate hook lateral radiographic view. Results of the study indicated that this 30 degrees -tilted lateral wrist radiographic projection with palmar abduction of the thumb clearly revealed the hamate hook from its base to the tip in the first web space away from the metacarpal bases and carpal bones. This radiographic technique was applied on patients who experienced pain at the hypothenar eminence. Fracture can be detected at the hamate hook in two cases and bilateral bipartite hamulus in one case. This specific supplementary projection is recommended in patients with the relevant physical signs.

A case of adenocarcinoma of the small intestine in a Japanese patient with Crohn disease: a report with immunohistochemical and oncogenic analyses.

We report a rare case of Crohn disease accompanied by a small-bowel carcinoma that developed in a 54-year-old Japanese man. The ulcerating tumor, which histologically proved to be a poorly differentiated adenocarcinoma and dysplasia surrounding the carcinoma, was located in the diseased ileum. The Ki-67 immunoreactive epithelial cells were increased in regenerative mucosa as compared with values for normal mucosa. The Ki-67- and p53-positive cells were increased in dysplasia and carcinoma as compared with values for regenerative or normal mucosa. In contrast, the p21(WAF1/CIP1) immunoreactive cells were decreased in this order. Intense DCC (deleted in colorectal cancer) expression was constantly shown among normal, regenerative, dysplastic and cancerous tissues. No bcl-2 expression and c-Ki-ras mutations were apparent. In conclusion, enhanced epithelial cell proliferation, p53 overexpression, and decrease of p21(WAF1/CIP1) expression may predispose the small-bowel mucosa to dysplasia and carcinoma development in Crohn disease.

Cell kinetics, p53 and bcl-2 expression, and c-Ki-ras mutations in flat-elevated tubulovillous adenomas and adenocarcinomas of the colorectum: comparison with polypoid lesions.

BACKGROUND: Flat(-elevated) tubulovillous adenomas and adenocarcinomas of the colorectum constitute a specific type of neoplasm with superficial spreading growth. To establish their characteristics, a comparative investigation of a series of tumors was performed. METHODS: A total of 56 flat tubulovillous tumors (39 adenomas, 17 invasive carcinomas) and 154 polypoid tubular or villous tumors (77 adenomas, 77 invasive carcinomas) were comparatively assessed for cell kinetics and molecular alterations. RESULTS: Ki-67 labeling and p53 expression for both types of tumors increased along with degree of dysplasia or invasion, whereas bcl-2 expression showed an inverse decrease. However, apoptotic activity was invariably low in the flat tubulovillous tumors, as compared with the polypoid tumors, in which increase was apparent with tumor progression. The flat tubulovillous tumors also showed a higher frequency of c-Ki-ras mutations (92.9%) than the polypoid tubular tumors (40.0%). CONCLUSIONS: The flat tubulovillous tumor can be considered a distinct entity, with characteristics different from the polypoid counterpart.

HLA-DR antigen and HLA-DRB1 genotyping with nonobstructive azoospermia in Japan.

We previously reported that the HLA-A33, -B13, and -B44 antigens, which are major histocompatibility complex class I molecules, are involved in the susceptibility of nonobstructive azoospermia in Japanese men. In this report, HLA-DR antigens, which are class II molecules, are investigated by advanced DNA typing in addition to classical serological typing to study a more complex genotype of HLA-DRB2. Genotyping was performed by the polymerase chain reaction-sequence-specific primer (PCR-SSP) method of analysis and/or by a commercial rapid assay based on the polymerase chain reaction (PCR), followed by reverse dot-blot hybridization of PCR products (the Inno-LiPA assay). The allele frequencies of the HLA-DR13 antigen and the -DRB1*1302 allele were significantly higher in Japanese subjects with nonobstructive azoospermia compared with a control group of healthy Japanese men, and these alleles were associated with relative risks for nonobstructive azoospermia of 4.2 and 4.9, respectively. If we suppose this strong linkage to both HLA class I and II antigens is due to linkage disequilibrium, it may suggest the existence of a novel gene involved in spermatogenesis in the class III region, which is located between the class I and class II regions and contains several genes other than HLA.