Analysis of Yersinia pseudotuberculosis Isolates Recovered from Deceased Mammals of a German Zoo Animal Collection.

Yersinia pseudotuberculosis is an important pathogen for both humans and animals. It can infect livestock, as well as pets and wild animals. During recent years, a number of reports have described the isolation of Y. pseudotuberculosis from zoo animals, mainly birds and mammals, for which the infection was mostly lethal. Between 2005 and 2019, there were at least 17 cases of deceased mammals, belonging to five different species, which suffered from a Y. pseudotuberculosis infection at the Zoo Wuppertal, Germany. Since only scarce information exists on the properties of Y. pseudotuberculosis from zoo animals, we characterized eight isolates, covering all infected species, in detail. All isolates were members of biotype 1, but belonged to five serotypes, five sequence types (STs), and seven core-genome multilocus sequence types (cgMLSTs). Using pulsed-field gel electrophoresis (PFGE) analysis and whole-genome sequencing (WGS), the seven isolates could be discriminated from each other. They differed significantly regarding their virulence genes and mobile genetic elements. While the virulence plasmid pYV existed in all serotypes (five isolates), a complete high-pathogenicity island (HPI) was detected only in the serotypes O:1a, O:1b, and O:13 (four isolates), but not in O:2a and O:2b. Similarly, the content of other plasmids and prophages varied greatly between the isolates. The data demonstrate that the deceased mammals were infected by seven individual isolates and not by a single type predominating in the zoo animals.

Reshaping preoperative treatment of pancreatic cancer in the era of precision medicine.

This review summarises the recent evidence on preoperative therapeutic strategies in pancreatic cancer and discusses the rationale for an imminent need for a personalised therapeutic approach in non-metastatic disease. The molecular diversity of pancreatic cancer and its influence on prognosis and treatment response, combined with the failure of 'all-comer' treatments to significantly impact on patient outcomes, requires a paradigm shift towards a genomic-driven approach. This is particularly important in the preoperative, potentially curable setting, where a personalised treatment allocation has the substantial potential to reduce pancreatic cancer mortality.

Gemcitabine-based adjuvant chemotherapy in subtypes of ampullary adenocarcinoma: international propensity score-matched cohort study.

BACKGROUND: Whether patients who undergo resection of ampullary adenocarcinoma have a survival benefit from adjuvant chemotherapy is currently unknown. The aim of this study was to compare survival between patients with and without adjuvant chemotherapy after resection of ampullary adenocarcinoma in a propensity score-matched analysis. METHODS: An international multicentre cohort study was conducted, including patients who underwent pancreatoduodenectomy for ampullary adenocarcinoma between 2006 and 2017, in 13 centres in six countries. Propensity scores were used to match patients who received adjuvant chemotherapy with those who did not, in the entire cohort and in two subgroups (pancreatobiliary/mixed and intestinal subtypes). Survival was assessed using the Kaplan-Meier method and Cox regression analyses. RESULTS: Overall, 1163 patients underwent pancreatoduodenectomy for ampullary adenocarcinoma. After excluding 187 patients, median survival in the remaining 976 patients was 67 (95 per cent c.i. 56 to 78) months. A total of 520 patients (53·3 per cent) received adjuvant chemotherapy. In a propensity score-matched cohort (194 patients in each group), survival was better among patients who received adjuvant chemotherapy than in those who did not (median survival not reached versus 60 months respectively; P = 0·051). A survival benefit was seen in patients with the pancreatobiliary/mixed subtype; median survival was not reached in patients receiving adjuvant chemotherapy and 32 months in the group without chemotherapy (P = 0·020). Patients with the intestinal subtype did not show any survival benefit from adjuvant chemotherapy. CONCLUSION: Patients with resected ampullary adenocarcinoma may benefit from gemcitabine-based adjuvant chemotherapy, but this effect may be reserved for those with the pancreatobiliary and/or mixed subtype.

ANTECEDENTES: Actualmente se desconoce si la quimioterapia adyuvante ofrece un beneficio en la supervivencia de los pacientes que se someten a resección de un adenocarcinoma ampular. El objetivo de este estudio fue comparar la supervivencia mediante la concordancia estimada por emparejamiento por puntaje de propensión, entre pacientes con y sin quimioterapia adyuvante después de la resección de un adenocarcinoma ampular. MÉTODOS: Se realizó un estudio internacional de cohortes multicéntrico, que incluyó a los pacientes que se sometieron a una duodenopancreatectomía por adenocarcinoma ampular (2006-2017) en 13 centros de seis países. Los puntajes de propensión se usaron para emparejar a los pacientes que recibieron quimioterapia adyuvante con los que no; tanto en la cohorte completa como en dos subgrupos (subtipo pancreaticobiliar / mixto e intestinal). La supervivencia se evaluó utilizando el método de Kaplan-Meier y las regresiones de Cox. RESULTADOS: En total, 1.163 pacientes fueron sometidos a una duodenopancreatectomía por adenocarcinoma ampular. Después de excluir a 179 pacientes, la mediana de supervivencia de los 976 pacientes restantes fue de 67 meses (i.c. del 95%, 56-78), de los cuales un total de 520 pacientes (53%) recibieron quimioterapia adyuvante. En una cohorte de emparejamiento por puntaje de propensión (194 versus 194 pacientes), la mediana de supervivencia fue mejor en los pacientes tratados con quimioterapia adyuvante en comparación con aquellos sin quimioterapia adyuvante (no se alcanzó la mediana de supervivencia versus 60 meses, respectivamente; P = 0,051). En el subtipo pancreaticobiliar/mixto se observó un beneficio en la supervivencia; no se alcanzó la mediana de supervivencia en pacientes que recibieron quimioterapia adyuvante versus 32 meses en el grupo sin quimioterapia, P = 0,020. El subtipo intestinal no mostró beneficio en la supervivencia de la quimioterapia adyuvante. CONCLUSIÓN: Los pacientes con adenocarcinoma ampular resecado pueden beneficiarse de la quimioterapia adyuvante basada en gemcitabina, pero este efecto podría reservarse para aquellos pacientes con subtipo de tumor pancreaticobiliar y/o mixto.

Optogenetic surface stimulation of the rat cervical spinal cord.

Electrical intraspinal microstimulation (ISMS) at various sites along the cervical spinal cord permits forelimb muscle activation, elicits complex limb movements and may enhance functional recovery after spinal cord injury. Here, we explore optogenetic spinal stimulation (OSS) as a less invasive and cell type-specific alternative to ISMS. To map forelimb muscle activation by OSS in rats, adeno-associated viruses (AAV) carrying the blue-light sensitive ion channels channelrhodopsin-2 (ChR2) and Chronos were injected into the cervical spinal cord at different depths and volumes. Following an AAV incubation period of several weeks, OSS-induced forelimb muscle activation and movements were assessed at 16 sites along the dorsal surface of the cervical spinal cord. Three distinct movement types were observed. We find that AAV injection volume and depth can be titrated to achieve OSS-based activation of several movements. Optical stimulation of the spinal cord is thus a promising method for dissecting the function of spinal circuitry and targeting therapies following injury. NEW & NOTEWORTHY Optogenetics in the spinal cord can be used both for therapeutic treatments and to uncover basic mechanisms of spinal cord physiology. For the first time, we describe the methodology and outcomes of optogenetic surface stimulation of the rat spinal cord. Specifically, we describe the evoked responses of forelimbs and address the effects of different adeno-associated virus injection paradigms. Additionally, we are the first to report on the limitations of light penetration through the rat spinal cord.

Defining the molecular pathology of pancreatic body and tail adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains a dismal disease, with very little improvement in survival over the past 50 years. Recent large-scale genomic studies have improved understanding of the genomic and transcriptomic landscape of the disease, yet very little is known about molecular heterogeneity according to tumour location in the pancreas; body and tail PDACs especially tend to have a significantly worse prognosis. The aim was to investigate the molecular differences between PDAC of the head and those of the body and tail of the pancreas. METHODS: Detailed correlative analysis of clinicopathological variables, including tumour location, genomic and transcriptomic data, was performed using the Australian Pancreatic Cancer Genome Initiative (APGI) cohort, part of the International Cancer Genome Consortium study. RESULTS: Clinicopathological data were available for 518 patients recruited to the APGI, of whom 421 underwent genomic analyses; 179 of these patients underwent whole-genome and 96 RNA sequencing. Patients with tumours of the body and tail had significantly worse survival than those with pancreatic head tumours (12·1 versus 22·0 months; P = 0·001). Location in the body and tail was associated with the squamous subtype of PDAC. Body and tail PDACs enriched for gene programmes involved in tumour invasion and epithelial-to-mesenchymal transition, as well as features of poor antitumour immune response. Whether this is due to a molecular predisposition from the outset, or reflects a later time point on the tumour molecular clock, requires further investigation using well designed prospective studies in pancreatic cancer. CONCLUSION: PDACs of the body and tail demonstrate aggressive tumour biology that may explain worse clinical outcomes.

Inter- and intra-tester reliability of clinical measurement to determine medio-lateral patellar position using a pachymeter or visual assessment.

The aim was to investigate inter-tester and intra-tester reliability and parallel reliability between a visual assessment method and a method using a pachymeter for locating the mid-point of the patella in determining the medial/lateral patella orientation. Fifteen asymptomatic subjects were assessed and the mid-point of the patella was determined by both methods on two separate occasions two weeks apart. Inter-tester reliability was obtained by ANOVA and by intraclass correlation coefficient (ICC); intra-tester reliability was obtained by a paired t-test and ICC; and parallel reliability was obtained by Pearson's Correlation and ICC, for the measurement on the first and second evaluations. There was acceptable inter-tester agreement (p=0.490) and reliability for the visual inspection (ICC=0.747) and for the pachymeter (ICC=0.716) at the second evaluation. The inter-tester reliability in the first evaluation was unacceptable (visual ICC=0.604; pachymeter ICC=0.612). Although there was statistical similarity between measurements for the first and second evaluations for all testers, intra-tester reliability was not acceptable for both methods: visual (examiner 1 ICC=0.175; examiner 2 ICC=0.189; examiner 3 ICC=0.155) and pachymeter (examiner 1 ICC=0.214; examiner 2 ICC=0.246; examiner 3 ICC=0.069). Parallel reliability gave a perfect correlation at the first evaluation (r=0.828; p<0.001) and at the second (r=0.756; p<0.001) and reliability was between acceptable and very good (ICC=[0.748-0.813]). Both visual and pachymeter methods provide reliable and similar medial/lateral patella orientation and are reliable between different examiners, but the results between the two assessments at 2 weeks' interval demonstrated an unacceptable reliability.

Preventive effects of perioperative parecoxib on post-discectomy pain.

BACKGROUND: Cyclooxygenase inhibitor treatment is viewed increasingly critical because of safety considerations, and there are several open questions on their optimal use. METHODS: In a randomized placebo-controlled study in 320 patients undergoing discectomy, we administered parecoxib 40 mg either perioperatively (before operation and after operation), after operation (first dose given in the evening after surgery), or before operation (single parecoxib dose given 45 min before surgery). We measured the main outcome variables: average pain score, morphine consumption, and opioid-related symptom distress at 25, 49, and 73 h after surgery. RESULTS: Perioperative parecoxib significantly (i) improved the pain score compared with both placebo and postoperative parecoxib, (ii) decreased morphine consumption, and (iii) reduced the opioid-related symptom distress score. Neither a single preoperative dose nor postoperative parecoxib (first dose given in the evening after surgery) significantly improved morphine's analgesic effectiveness. CONCLUSIONS: Perioperative parecoxib compared with postoperative parecoxib improves post-discectomy pain and results in a reduction in adverse effects associated with opioid therapy. Postoperative parecoxib, or a single pre-incisional parecoxib dose, does not significantly improve post-discectomy pain or opioid side-effects up to 3 days after surgery.

Regulation of glomerular basement membrane collagen expression by LMX1B contributes to renal disease in nail patella syndrome.

Basement membrane (BM) morphogenesis is critical for normal kidney function. Heterotrimeric type IV collagen, composed of different combinations of six alpha-chains (1-6), is a major matrix component of all BMs (ref. 2). Unlike in other BMs, glomerular BM (GBM) contains primarily the alpha 3(IV) and alpha 4(IV) chains, together with the alpha 5(IV) chain. A poorly understood, coordinated temporal and spatial switch in gene expression from ubiquitously expressed alpha 1(IV) and alpha 2(IV) collagen to the alpha 3(IV), alpha 4(IV) and alpha 5(IV) chains occurs during normal embryogenesis of GBM (ref. 4). Structural abnormalities of type IV collagen have been associated with diverse biological processes including defects in molecular filtration in Alport syndrome, cell differentiation in hereditary leiomyomatosis, and autoimmunity in Goodpasture syndrome; however, the transcriptional and developmental regulation of type IV collagen expression is unknown. Nail patella syndrome (NPS) is caused by mutations in LMX1B, encoding a LIM homeodomain transcription factor. Some patients have nephrosis-associated renal disease characterized by typical ultrastructural abnormalities of GBM (refs. 8,9). In Lmx1b(-/-) mice, expression of both alpha(3)IV and alpha(4)IV collagen is strongly diminished in GBM, whereas that of alpha1, alpha2 and alpha5(IV) collagen is unchanged. Moreover, LMX1B binds specifically to a putative enhancer sequence in intron 1 of both mouse and human COL4A4 and upregulates reporter constructs containing this enhancer-like sequence. These data indicate that LMX1B directly regulates the coordinated expression of alpha 3(IV) and alpha 4(IV) collagen required for normal GBM morphogenesis and that its dysregulation in GBM contributes to the renal pathology and nephrosis in NPS.

LMX1B transactivation and expression in nail-patella syndrome.

Lmx1b, a member of the LIM homeodomain protein family, is essential for the specification of dorsal limb fates at the zeugopodal and autopodal level in vertebrates. We and others have shown that a skeletal dysplasia, nail-patella syndrome (NPS), results from mutations in LMX1B. While it is a unique mesenchymal determinant of dorsal limb patterning during vertebrate development, the mechanism by which LMX1B mutations generate the NPS phenotype has not been addressed at a transcriptional level or correlated with its spatial pattern of gene expression. In this study, in situ hybridizations of Lmx1b on murine limb sections reveal strong expression in dorsal mesenchymal tissues (precursors of muscle, tendons, joints and patella) and, interestingly, also in anterior structures of the limb, explaining the anterior to posterior gradient of joint and nail dysplasia observed in NPS patients. Transfection studies showed that both the LIM domain-interacting protein, LDB1, and the helix-loop-helix protein, E47/shPan1, can regulate LMX1B action. While co--transfections of E47/shPan1 with LMX1B result in a synergistic effect on reporter activity, LDB1 down-regulated LMX1B-mediated transactivation irrespective of E47/shPan1. Mutant LMX1B proteins containing human mutations affecting each of the helices or the N-terminal arm of the homeodomain abolished transactivation, while LIM B and truncation mutations retained residual activity. These mutations fail to act in a dominant-negative manner on wild-type LMX1B in mixing studies, thereby supporting haploinsufficiency as the mechanism underlying NPS pathogenesis.

Isolation, characterization, and mapping of a zinc finger gene, ZFP95, containing both a SCAN box and an alternatively spliced KRAB A domain.

A new zinc finger gene of the Krüppel family was identified by screening a human fetal cartilage cDNA library with degenerate oligonucleotides. Sequence analysis indicates that ZFP95 contains 12 highly conserved zinc finger motifs at the C-terminus and a SCAN box as well as a KRAB A domain at the N-terminus of the protein. ZFP95 represents a member of a new subclass of Krüppel zinc finger proteins containing both a SCAN box and a KRAB domain. Sequence comparison revealed that ZFP95 is the human ortholog of murine Zfp95, which is differentially expressed during spermatogenesis. We demonstrate that ZFP95 is ubiquitously expressed in adult and fetal tissues with the strongest expression in testis. Two transcripts, 4. 2 and 4.6 kb, were detected in all tissues tested. In testis, a third transcript of 3.8 kb was present. RT-PCR analysis confirmed alternative splicing for the KRAB A domain and an upstream exon leading to three transcripts of ZFP95 with and without this transcriptional repressor domain. Finally, we show that ZFP95 maps on human chromosome 7q22 between the markers D7S651 and WI-5853.

Identification of LMX1B gene point mutations in italian patients affected with Nail-Patella syndrome.

Nail-Patella syndrome, or osteo-onychodysplasia, is an autosomal dominant disorder characterized by nail dysplasia, absent or hypoplastic patellae, iliac horns and nephropathy. Previous studies have demonstrated linkage of the Nail-Patella locus with polymorphic markers on human chromosome 9q34. Recently, point mutations in the LMX1B gene have been identified in Nail-Patella patients and in families with recurrence of Nail-Patella syndrome and open-angle glaucoma. We describe here the identification of additional point mutations in the LMX1B gene in a set of Italian patients affected with Nail-Patella syndrome: two deletions of 1 and 2 bp causing a frameshift in two sporadic patients and nonsense mutations in two familial and one sporadic cases have been identified. All the mutations affect the homeodomain of the LMX1B protein and could cause the Nail-Patella syndrome through a loss of function as well as a dominant negative effect. Haplotype analysis in the two familial cases carrying the same stop codon mutation suggests the presence of a founder effect. Finally, analysis of cDNA clones obtained from human fetal kidney has revealed the existence of two different transcripts of LMX1B gene likely due to an alternative splicing.

Natural history of rheumatoid arthritis of the cervical spine.

This article reviews the natural history of rheumatoid arthritis involving the cervical spine with special attention given to predictors of paralysis. Understanding the natural history of rheumatoid arthritis of the cervical spine is necessary to determine the benefit of various interventions. The primary treatment goal for cervical instability is prevention of irreversible neurologic injury. The natural history of rheumatoid arthritis for a period of 10 years or more is one of significant disease progression. The natural history of cervical instability in patients with rheumatoid arthritis is more variable, with only some patients having a neurologic deficit develop. Recent studies support prophylactic stabilization of the rheumatoid cervical spine to prevent paralysis in high risk patients. However, proponents for prophylactic arthrodesis must acknowledge that not all cervical instability in rheumatoid arthritis progresses to neurologic deficit, and surgical intervention in patients with rheumatoid arthritis incurs added morbidity and mortality. Identifying the risk factors for progression of cervical instability is the first step in eliminating morbidity and mortality from spinal cord and brain stem compression. Surgical stabilization is indicated not only for those patients with paralysis, but for the subgroups of patients with cervical rheumatoid disease who are at risk for spinal cord and brain stem compression. The posterior atlantodental interval is the most reliable screening tool and predictor of progressive neurologic deficit.

Mutation analysis of LMX1B gene in nail-patella syndrome patients.

Nail-patella syndrome (NPS), a pleiotropic disorder exhibiting autosomal dominant inheritance, has been studied for >100 years. Recent evidence shows that NPS is the result of mutations in the LIM-homeodomain gene LMX1B. To determine whether specific LMX1B mutations are associated with different aspects of the NPS phenotype, we screened a cohort of 41 NPS families for LMX1B mutations. A total of 25 mutations were identified in 37 families. The nature of the mutations supports the hypothesis that NPS is the result of haploinsufficiency for LMX1B. There was no evidence of correlation between aspects of the NPS phenotype and specific mutations.

Malignant tumors of the kidney, brain, and soft tissues in children and young adults with the tuberous sclerosis complex.

BACKGROUND: The tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation, and benign tumors of the brain, heart, skin, and kidney. Malignant tumors also can occur in patients with tuberous sclerosis, particularly in the kidney, although they occur less frequently than benign tumors. The types of malignancy that occur in TSC have not been characterized fully. METHODS: Clinical and pathologic features of 8 malignant tumors from 6 TSC patients ranging in age from 22 months to 21 years are reviewed. Six tumors were renal, one was from the inguinal region, and one was from the brain. The tumors were analyzed for loss of heterozygosity (LOH) in the chromosomal regions of the TSC1, TSC2, and VHL genes. RESULTS: Three patients (ages 7, 8, and 20 years) had renal cell carcinomas (RCCs). Two of these patients had multifocal RCCs. All three patients with RCC also had prominent multifocal dysplasia of renal cyst epithelium. Two patients (ages 20 and 21 years) had malignant angiomyolipomas (1 renal and 1 inguinal). One patient (age 22 months) had a Grade 4 giant cell astrocytoma (glioblastoma multiforme). LOH in the region of the TSC2 gene was found, either in the malignant tumor or in benign tumors, in all five patients whose DNA could be analyzed. CONCLUSIONS: Children with TSC, as well as adults with the disease, are at risk for developing malignant tumors. Two types of renal malignancy occur in TSC: RCC, which appears to arise from dysplastic renal cyst epithelial cells, and malignant angiomyolipoma. Tumors cytologically similar to malignant angiomyolipomas also may occur at extrarenal sites. LOH analyses suggest that the majority of patients with TSC who develop malignant tumors have germline TSC2, rather than TSC1, gene mutations.

The centromeric/nucleolar chromatin protein ZFP-37 may function to specify neuronal nuclear domains.

Murine ZFP-37 is a member of the large family of C2H2 type zinc finger proteins. It is characterized by a truncated NH2-terminal Krüppel-associated box and is thought to play a role in transcriptional regulation. During development Zfp-37 mRNA is most abundant in the developing central nervous system, and in the adult mouse expression is restricted largely to testis and brain. Here we show that at the protein level ZFP-37 is detected readily in neurons of the adult central nervous system but hardly in testis. In brain ZFP-37 is associated with nucleoli and appears to contact heterochromatin. Mouse and human ZFP-37 have a basic histone H1-like linker domain, located between KRAB and zinc finger regions, which binds double-stranded DNA. Thus we suggest that ZFP-37 is a structural protein of the neuronal nucleus which plays a role in the maintenance of specialized chromatin domains.

Cloning, characterization, and chromosomal assignment of the human ortholog of murine Zfp-37, a candidate gene for Nager syndrome.

In an effort to identify putative transcription factors involved in chondrocyte differentiation during human endochondral bone formation, a human fetal cartilage-specific cDNA library was screened with a degenerate oligonucleotide probe corresponding to a conserved stretch of eight amino acids from the zinc finger region of the Drosophila Krüppel gene family of DNA-binding proteins. Using this strategy, we have identified a novel zinc finger gene ZFP-37. ZFP-37 corresponds to a putative transcription factor containing 12 tandemly repeated zinc finger motifs and a Krüppel-associated box (KRAB) domain. The KRAB domain has been reported to function as a transcriptional repressor and is located in the amino terminus, while the zinc finger repeats are positioned at the carboxy-terminal end of ZFP-37. Gene mapping with a somatic cell hybrid panel and fluorescence in situ hybridization (FISH) localized ZFP-37 to human Chr 9q32. The gene is expressed at low level as a 3.2-kb mRNA in several tissues including fetal human cartilage. Sequence comparison revealed that ZFP-37 may represent the human homolog of the mouse gene Zfp-37. The map location and expression pattern suggest ZFP-37 as a candidate gene for a craniofacial-limb malformation, Nager syndrome (acrofacial dysostosis).

Mutations in LMX1B cause abnormal skeletal patterning and renal dysplasia in nail patella syndrome.

The LIM-homeodomain protein Lmx1b plays a central role in dorso-ventral patterning of the vertebrate limb. Targeted disruption of Lmx1b results in skeletal defects including hypoplastic nails, absent patellae and a unique form of renal dysplasia (see accompanying manuscript by H. Chen et al.; ref. 2). These features are reminiscent of the dominantly inherited skeletal malformation nail patella syndrome (NPS). We show that LMX1B maps to the NPS locus and that three independent NPS patients carry de novo heterozygous mutations in this gene. Functional studies show that one of these mutations disrupts sequence-specific DNA binding, while the other two mutations result in premature termination of translation. These data demonstrate a unique role for LMX1B in renal development and in patterning of the skeletal system, and suggest that alteration of Lmx1b/LMX1B function in mice and humans results in similar phenotypes. Furthermore, we provide evidence for the first described mutations in a LIM-homeodomain protein which account for an inherited form of abnormal skeletal patterning and renal failure.