Large Community Outbreak of Legionnaires Disease Potentially Associated with a Cooling Tower - Napa County, California, 2022.

Legionnaires disease is a serious infection acquired by inhalation of water droplets from human-made building water systems that contain Legionella bacteria. On July 11 and 12, 2022, Napa County Public Health (NCPH) in California received reports of three positive urinary antigen tests for Legionella pneumophila serogroup 1 in the town of Napa. By July 21, six Legionnaires disease cases had been confirmed among Napa County residents, compared with a baseline of one or two cases per year. NCPH requested assistance from the California Department of Public Health (CDPH) and CDC to aid in the investigations. Close temporal and geospatial clustering permitted a focused environmental sampling strategy of high-risk facilities which, coupled with whole genome sequencing results from samples and investigation of water system maintenance, facilitated potential linking of the outbreak with an environmental source. NCPH, with technical support from CDC and CDPH, instructed and monitored remediation practices for all environmental locations that tested positive for Legionella. The investigation response to this community outbreak illustrates the importance of interdisciplinary collaboration by public health agencies, laboratory support, timely communication with the public, and cooperation of managers of potentially implicated water systems. Timely identification of possible sources, sampling, and remediation of any facility testing positive for Legionella is crucial to interrupting further transmission.

Multiplex Real-time PCR Assay for the Detection of all Chlamydia Species and Simultaneous Differentiation of C. psittaci and C. pneumoniae in Human Clinical Specimens.

We developed and assessed the performance of a new multiplex real-time PCR assay for the detection of all Chlamydia species and simultaneous differentiation of Chlamydia psittaci and Chlamydia pneumoniae-two important human respiratory pathogens-in human clinical specimens. Next-generation sequencing was used to identify unique targets to design real-time PCR assays targeting all Chlamydia species, C. psittaci, and C. pneumoniae. To validate the assay, we used a panel of 49 culture isolates comprising seven C. psittaci genotypes, eight C. pneumoniae isolates, seven other Chlamydia species, and 22 near-neighbor bacterial and viral isolates, along with 22 specimens from external quality assessment (EQA) panels and 34 nasopharyngeal and oropharyngeal swabs and cerebrospinal fluid, stool, and sputum specimens previously identified as positive or negative for C. psittaci or C. pneumoniae. The assays were 100% specific, with limits of detection of 7.64- 9.02 fg/µL. The assay results matched with historical assay results for all specimens, except for one owing to the increased sensitivity of the new C. psittaci assay; the results of the EQA specimens were 100% accurate. This assay may improve the timely and accurate clinical diagnosis of Chlamydia infections and provide a greater understanding of the burden of disease caused by these agents.

Axin phosphorylation in both Wnt-off and Wnt-on states requires the tumor suppressor APC.

The aberrant activation of Wnt signal transduction initiates the development of 90% of colorectal cancers, the majority of which arise from inactivation of the tumor suppressor Adenomatous polyposis coli (APC). In the classical model for Wnt signaling, the primary role of APC is to act, together with the concentration-limiting scaffold protein Axin, in a "destruction complex" that directs the phosphorylation and consequent proteasomal degradation of the transcriptional activator ß-catenin, thereby preventing signaling in the Wnt-off state. Following Wnt stimulation, Axin is recruited to a multiprotein "signalosome" required for pathway activation. Whereas it is well-documented that APC is essential in the destruction complex, APC's role in this complex remains elusive. Here, we demonstrate in Drosophila that Axin exists in two distinct phosphorylation states in Wnt-off and Wnt-on conditions, respectively, that underlie its roles in the destruction complex and signalosome. These two Axin phosphorylation states are catalyzed by glycogen synthase kinase 3 (GSK3), and unexpectedly, completely dependent on APC in both unstimulated and Wnt-stimulated conditions. In a major revision of the classical model, we show that APC is essential not only in the destruction complex, but also for the rapid transition in Axin that occurs after Wnt stimulation and Axin's subsequent association with the Wnt co-receptor LRP6/Arrow, one of the earliest steps in pathway activation. We propose that this novel requirement for APC in Axin regulation through phosphorylation both prevents signaling in the Wnt-off state and promotes signaling immediately following Wnt stimulation.

Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling.

The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, attenuating and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor adenomatous polyposis coli or casein kinase 1α uncovered new regulatory features at most levels of the pathway. These include a requirement for the transcription factor AP-4, a role for the DAX domain of AXIN2 in controlling ß-catenin transcriptional activity, a contribution of glycophosphatidylinositol anchor biosynthesis and glypicans to R-spondin-potentiated WNT signaling, and two different mechanisms that regulate signaling when distinct components of the ß-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems.

Dual Roles for Membrane Association of Drosophila Axin in Wnt Signaling.

Deregulation of the Wnt signal transduction pathway underlies numerous congenital disorders and cancers. Axin, a concentration-limiting scaffold protein, facilitates assembly of a "destruction complex" that prevents signaling in the unstimulated state and a plasma membrane-associated "signalosome" that activates signaling following Wnt stimulation. In the classical model, Axin is cytoplasmic under basal conditions, but relocates to the cell membrane after Wnt exposure; however, due to the very low levels of endogenous Axin, this model is based largely on examination of Axin at supraphysiological levels. Here, we analyze the subcellular distribution of endogenous Drosophila Axin in vivo and find that a pool of Axin localizes to cell membrane proximal puncta even in the absence of Wnt stimulation. Axin localization in these puncta is dependent on the destruction complex component Adenomatous polyposis coli (Apc). In the unstimulated state, the membrane association of Axin increases its Tankyrase-dependent ADP-ribosylation and consequent proteasomal degradation to control its basal levels. Furthermore, Wnt stimulation does not result in a bulk redistribution of Axin from cytoplasmic to membrane pools, but causes an initial increase of Axin in both of these pools, with concomitant changes in two post-translational modifications, followed by Axin proteolysis hours later. Finally, the ADP-ribosylated Axin that increases rapidly following Wnt stimulation is membrane associated. We conclude that even in the unstimulated state, a pool of Axin forms membrane-proximal puncta that are dependent on Apc, and that membrane association regulates both Axin levels and Axin's role in the rapid activation of signaling that follows Wnt exposure.

The ADP-ribose polymerase Tankyrase regulates adult intestinal stem cell proliferation during homeostasis in Drosophila.

Wnt/ß-catenin signaling controls intestinal stem cell (ISC) proliferation, and is aberrantly activated in colorectal cancer. Inhibitors of the ADP-ribose polymerase Tankyrase (Tnks) have become lead therapeutic candidates for Wnt-driven cancers, following the recent discovery that Tnks targets Axin, a negative regulator of Wnt signaling, for proteolysis. Initial reports indicated that Tnks is important for Wnt pathway activation in cultured human cell lines. However, the requirement for Tnks in physiological settings has been less clear, as subsequent studies in mice, fish and flies suggested that Tnks was either entirely dispensable for Wnt-dependent processes in vivo, or alternatively, had tissue-specific roles. Here, using null alleles, we demonstrate that the regulation of Axin by the highly conserved Drosophila Tnks homolog is essential for the control of ISC proliferation. Furthermore, in the adult intestine, where activity of the Wingless pathway is graded and peaks at each compartmental boundary, Tnks is dispensable for signaling in regions where pathway activity is high, but essential where pathway activity is relatively low. Finally, as observed previously for Wingless pathway components, Tnks activity in absorptive enterocytes controls the proliferation of neighboring ISCs non-autonomously by regulating JAK/STAT signaling. These findings reveal the requirement for Tnks in the control of ISC proliferation and suggest an essential role in the amplification of Wnt signaling, with relevance for development, homeostasis and cancer.

Wnt pathway activation by ADP-ribosylation.

Wnt/ß-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)--known to target Axin for proteolysis-regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly.

Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase.

Wnt/ß-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multiprotein "destruction complex," assembled by the scaffold protein Axin, targets the key transcriptional activator ß-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. Here, we use an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. We find that for nearly all Wingless-driven developmental processes, a three- to fourfold increase in Axin is insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, we find that both the tumor suppressor adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and we define separable domains in Axin that are important for APC- or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation.

Toggling a conformational switch in Wnt/ß-catenin signaling: regulation of Axin phosphorylation. The phosphorylation state of Axin controls its scaffold function in two Wnt pathway protein complexes.

The precise orchestration of two opposing protein complexes - one in the cytoplasm (ß-catenin destruction complex) and the other at the plasma membrane (LRP6 signaling complex) - is critical for controlling levels of the transcriptional co-factor ß-catenin, and subsequent activation of the Wnt/ß-catenin signal transduction pathway. The Wnt pathway component Axin acts as an essential scaffold for the assembly of both complexes. How the ß-catenin destruction and LRP6 signaling complexes are modulated following Wnt stimulation remains controversial. A recent study in Science by He and coworkers reveals an underlying logic for Wnt pathway control in which Axin phosphorylation toggles a switch between the active and inactive states. This mini-review focuses on this and two other recent studies that provide insight into the initial signaling events triggered by Wnt exposure. We emphasize regulation of the ß-catenin destruction and LRP6 signaling complexes and propose a framework for future work in this area.

Norepinephrine deficiency is caused by combined abnormal mRNA processing and defective protein trafficking of dopamine beta-hydroxylase.

Human norepinephrine (NE) deficiency (or dopamine ß-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations, such as a common splice donor site mutation (IVS1+2T→C) and various missense mutations, in NE deficiency patients were identified, molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T→C mutation results in a non-detectable level of DBH protein production and that all three missense mutations tested lead to the DBH protein being trapped in the endoplasmic reticulum (ER). Supporting the view that mutant DBH induces an ER stress response, exogenous expression of mutant DBH dramatically induced expression of BiP, a master ER chaperone. Furthermore, we found that a pharmacological chaperone, glycerol, significantly rescued defective trafficking of mutant DBH proteins. Taken together, we propose that NE deficiency is caused by the combined abnormal processing of DBH mRNA and defective protein trafficking and that this disease could be treated by a pharmacological chaperone(s).

Direct reprogramming of rat neural precursor cells and fibroblasts into pluripotent stem cells.

BACKGROUND: Given the usefulness of rats as an experimental system, an efficient method for generating rat induced pluripotent stem (iPS) cells would provide researchers with a powerful tool for studying human physiology and disease. Here, we report direct reprogramming of rat neural precursor (NP) cells and rat embryonic fibroblasts (REF) into iPS cells by retroviral transduction using either three (Oct3/4, Sox2, and Klf4), four (Oct3/4, Sox2, Klf4, and c-Myc), or five (Oct3/4, Sox2, Klf4, c-Myc, and Nanog) genes. METHODOLOGY AND PRINCIPAL FINDINGS: iPS cells were generated from both NP and REF using only three (Oct3/4, Sox2, and Klf4) genes without c-Myc. Two factors were found to be critical for efficient derivation and maintenance of rat iPS cells: the use of rat instead of mouse feeders, and the use of small molecules specifically inhibiting mitogen-activated protein kinase and glycogen synthase kinase 3 pathways. In contrast, introduction of embryonic stem cell (ESC) extracts induced partial reprogramming, but failed to generate iPS cells. However, when combined with retroviral transduction, this method generated iPS cells with significantly higher efficiency. Morphology, gene expression, and epigenetic status confirmed that these rat iPS cells exhibited ESC-like properties, including the ability to differentiate into all three germ layers both in vitro and in teratomas. In particular, we found that these rat iPS cells could differentiate to midbrain-like dopamine neurons with a high efficiency. CONCLUSIONS/SIGNIFICANCE: Given the usefulness of rats as an experimental system, our optimized method would be useful for generating rat iPS cells from diverse tissues and provide researchers with a powerful tool for studying human physiology and disease.

Differential gene expression profiles of metastases in paired primary and metastatic colorectal carcinomas.

BACKGROUND AND METHODS: Despite the overwhelming clinical significance of metastases, the cellular and molecular mechanisms involved are largely unknown. In order to define significant differences between primary colon carcinomas and their metastases, we analyzed gene expression profiles of 12 sets of triple-paired tissues using 19 K human oligonucleotide microarrays. A total of 36 microarray experiments were analyzed by unsupervised two-way hierarchical clustering and multi-dimensional scaling (MDS). RESULTS: Both methods completely distinguished normal mucosa from carcinoma, but failed to demonstrate a complete classification of primary and metastatic carcinomas. We found a separable tendency to be classified into the primary and metastatic colon carcinomas by MDS. In supervised hierarchical clustering, we identified 80 genes that were differentially expressed between paired primary and metastatic colon carcinomas. The 80 identified genes also successfully distinguished three validation sets of primary and lung-metastatic colon carcinomas. A specific set of genes was identified that distinguished the metastasis from the corresponding primary tumor in nearly half of the metastases analyzed. CONCLUSIONS: We suggest that a more accurate model of the metastatic potential is based on a global tumor expression pattern along with the appearance of distinct metastatic variants. This molecular profiling may be useful for the future study of colon cancer metastasis.

Frequent inactivation of SPARC by promoter hypermethylation in colon cancers.

Epigenetic modification of gene expression plays an important role in the development of human cancers. The inactivation of SPARC through CpG island methylation was studied in colon cancers using oligonucleotide microarray analysis and methylation specific PCR (MSP). Gene expression of 7 colon cancer cell lines was evaluated before and after treatment with the demethylating agent 5-aza-2'-deoxycytidine (5Aza-dC) by oligonucleotide microarray analysis. Expression of SPARC was further examined in colon cancer cell lines and primary colorectal cancers, and the methylation status of the SPARC promoter was determined by MSP. SPARC expression was undetectable in 5 of 7 (71%) colorectal cancer cell lines. Induction of SPARC was demonstrated after treatment with the demethylating agent 5Aza-dC in 5 of the 7 cell lines. We examined the methylation status of the CpG island of SPARC in 7 colon cancer cell lines and in 20 test set of colon cancer tissues. MSP demonstrated hypermethylation of the CpG island of SPARC in 6 of 7 cell lines and in all 20 primary colon cancers, when compared with only 3 of 20 normal colon mucosa. Immunohistochemical analysis showed that SPARC expression was downregulated or absent in 17 of 20 colon cancers. A survival analysis of 292 validation set of colorectal carcinoma patients revealed a poorer prognosis for patients lacking SPARC expression than for patients with normal SPARC expression (56.79% vs. 75.83% 5-year survival rate, p = 0.0014). The results indicate that epigenetic gene silencing of SPARC is frequent in colon cancers, and that inactivation of SPARC is related to rapid progression of colon cancers.

Differentially expressed proteins in gastrointestinal stromal tumors with KIT and PDGFRA mutations.

Most gastrointestinal stromal tumors (GIST) have activating mutations in either KIT or PDGFRA. However, a small subset of GIST lacks either mutation. To investigate the molecular characteristics of GIST according to mutation type, protein expression profiles in 12 GIST (2 cases with PDGFRA mutations, 8 cases with KIT mutations and 2 cases lacking either mutation) were analyzed using 2-DE and MALDI-TOF-MS. Comparative analysis of the respective spot patterns using 2-DE showed that 15 proteins were differently expressed according to the mutation status. Expression levels of septin and heat shock protein (HSP) 27 were increased in GIST with KIT mutations and annexin V was overexpressed in GIST lacking either mutation. Among the 15 proteins, overexpression of 5 proteins [annexin V, high mobility group protein 1 (HMGB1), C13orf2, glutamate dehydrogenase 1 and fibrinogen beta chain] and decreased expression of RoXaN correlated with a higher tumor grade. These findings suggest that differential protein expression can be used as a diagnostic biomarker. Moreover, it may play a role in the development and progression of GIST according to activating mutation type, as these proteins have been shown to be involved in tumor metastasis, apoptosis and immune response.

Impaired nonhomologous end-joining in mismatch repair-deficient colon carcinomas.

Frameshift mutations of coding mononucleotide repeat of the hRAD50 gene and formation of the mutant hMRE11 splicing variant are frequent events in tumors with mismatch repair (MMR) deficiency. Both the hRAD50 and hMRE11 proteins form a heterotrimer with the NBS1, and this heterotrimer is involved in the double strand DNA break repair by homologous recombination and nonhomologous end-joining (NHEJ). In order to clarify the role of hRAD50 and hMRE11 gene alterations in MMR-deficient tumors, we analyzed the expression of the hRAD50 and hMRE11 proteins and we evaluated NHEJ in the seven MMR-deficient and five MMR-proficient colon cancer cell lines. Frameshift mutations of the hRAD50 gene were found in five of seven MMR-deficient cell lines, and this was directly related to the decreased expression of hRAD50 mRNA and protein. The mutant hMRE11 splicing variant was found in all of the seven MMR-deficient cell lines, and this was related to the decreased hMRE11 expression in four of the seven MMR-deficient cell lines. MMR-deficient cell lines with decreased hRAD50 and hMRE11 expressions were more sensitive to gamma-irradiation, and these cell lines showed an impaired NHEJ. The impairment of NHEJ was induced after knockdown of hRAD50 and hMRE11 through small interference RNA. Our findings suggest that mutations of hRAD50 and hMRE11 genes in MMR-deficient tumors are related to the defects in NHEJ, and this may result in chromosomal changes during the progression of tumor.