Colon tumour cell death causes mTOR dependence by paracrine P2X4 stimulation.

Solid cancers exhibit a dynamic balance between cell death and proliferation ensuring continuous tumour maintenance and growth1,2. Increasing evidence links enhanced cancer cell apoptosis to paracrine activation of cells in the tumour microenvironment initiating tissue repair programs that support tumour growth3,4, yet the direct effects of dying cancer cells on neighbouring tumour epithelia and how this paracrine effect potentially contributes to therapy resistance are unclear. Here we demonstrate that chemotherapy-induced tumour cell death in patient-derived colorectal tumour organoids causes ATP release triggering P2X4 (also known as P2RX4) to mediate an mTOR-dependent pro-survival program in neighbouring cancer cells, which renders surviving tumour epithelia sensitive to mTOR inhibition. The induced mTOR addiction in persisting epithelial cells is due to elevated production of reactive oxygen species and subsequent increased DNA damage in response to the death of neighbouring cells. Accordingly, inhibition of the P2X4 receptor or direct mTOR blockade prevents induction of S6 phosphorylation and synergizes with chemotherapy to cause massive cell death induced by reactive oxygen species and marked tumour regression that is not seen when individually applied. Conversely, scavenging of reactive oxygen species prevents cancer cells from becoming reliant on mTOR activation. Collectively, our findings show that dying cancer cells establish a new dependency on anti-apoptotic programs in their surviving neighbours, thereby creating an opportunity for combination therapy in P2X4-expressing epithelial tumours.

Pet dogs (Canis familiaris) re-engage humans after joint activity.

Joint intentionality, the mutual understanding of shared goals or actions to partake in a common task, is considered an essential building block of theory of mind in humans. Domesticated dogs are unusually adept at comprehending human social cues and cooperating with humans, making it possible that they possess behavioral signatures of joint intentionality in interactions with humans. Horschler and colleagues (Anim Behav 183: 159-168, 2022) examined joint intentionality in a service dog population, finding that upon interruption of a joint experience, dogs preferentially re-engaged their former partner over a passive bystander, a behavior argued to be a signature of joint intentionality in human children. In the current study, we aimed to replicate and extend these results in pet dogs. One familiar person played with the dog and then abruptly stopped. We examined if dogs would preferentially re-engage the player instead of a familiar bystander who was also present. Consistent with the findings of Horschler and colleagues (Anim Behav 183: 159-168, 2022), pet dogs preferentially gazed toward and offered the toy to the player significantly more than the familiar bystander. However, no difference was observed in physical contact. These findings provide preliminary evidence for behavioral signatures of joint intentionality in pet dogs, but future work is needed to understand whether this phenomenon extends to other contexts.

Predictors of Mortality and Revision Following Digital Amputation for Infection and Necrosis.

PURPOSE: Digital amputation is a commonly performed procedure for infection and necrosis in patients with diabetes, peripheral vascular disease (PVD), and on dialysis. There is a lack of data regarding prognosis for revision amputation and mortality following digital amputation in these patients. METHODS: All digital amputations over 10-year period (2008-2018) at a single center were reviewed. There were 484 amputations in 360 patients, among which 358 were performed for trauma (reference sample) and 126 for infection or necrosis (sample of interest). Patient death and revision were determined from National Vital Statistics System and medical records. Propensity score matching was performed to compare groups. Data were then compared to the Social Security Administration Actuarial Life Table for 2015 to determine age-matched expected mortality. RESULTS: The 2-year revision rate was 34% for amputations performed for infection or necrosis, compared to 15% for amputations due to trauma. For amputations performed for infection or necrosis, the revision rate was 47.7% when diabetes, PVD, and dialysis were present. Among all patients with infection or necrosis (n = 104) undergoing a digital amputation, overall survival at 2, 5, and 10 years was 79.4%, 57.3%, and 17.5%, respectively, which represented a 3.2-fold increased risk of death compared to controls. (hazard ratio, 3.19; 95% confidence interval, 1.47-6.93). For amputations due to trauma, mortality was no different from that in the age-matched general population. CONCLUSIONS: Mortality and revision risk are high for patients requiring a digital amputation for infection or necrosis and are further increased with medical comorbidities. Hand surgeons should consider the prognostic implications of these data when counseling patients. TYPE OF STUDY/LEVEL OF EVIDENCE: Prognostic IV.

The effect of operative time on surgical-site infection following total shoulder arthroplasty.

BACKGROUND: Many factors contribute to the risk of surgical-site infection (SSI) following total shoulder arthroplasty (TSA). Operative time is a modifiable factor that may contribute to SSI occurrence after TSA. This study aimed to determine the correlation between operative time and SSI following TSA. MATERIALS AND METHODS: By use of the American College of Surgeons National Surgical Quality Improvement Program database, a total of 33,987 patient records were queried from 2006 to 2020 and sorted by operative time and the development of an SSI in the 30-day postoperative period. Odds ratios for the development of an SSI were calculated based on operative time. RESULTS: An SSI developed in the 30-day postoperative period in 169 of the 33,470 patients in this study, resulting in an overall SSI rate of 0.50%. A positive correlation was identified between operative time and the SSI rate. An inflection point was identified at an operative time of 180 minutes, with a significant increase in the rate of SSI occurrence for operative times >180 minutes. DISCUSSION AND CONCLUSION: Increased operative time was shown to be strongly correlated with an increased risk of SSI within 30 days following surgery, with a significant inflection point at 180 minutes. The target operative time for TSA should be <180 minutes to reduce the risk of SSI.

Low Fuel Convergence Path to Direct-Drive Fusion Ignition.

A new class of inertial fusion capsules is presented that combines multishell targets with laser direct drive at low intensity (2.8×10^{14} W/cm^{2}) to achieve robust ignition. The targets consist of three concentric, heavy, metal shells, enclosing a volume of tens of µg of liquid deuterium-tritium fuel. Ignition is designed to occur well "upstream" from stagnation, with minimal pusher deceleration to mitigate interface Rayleigh-Taylor growth. Laser intensities below thresholds for laser plasma instability and cross beam energy transfer facilitate high hydrodynamic efficiency (∼10%).

Non-stem cell lineages as an alternative origin of intestinal tumorigenesis in the context of inflammation.

According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, has been shown to suppress intestinal stemness. Here, we used Paneth cells as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation in mice. Upon inflammation, Paneth cell-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in patients with inflammatory bowel disease, but also of a larger fraction of human sporadic colon cancers. The latter is possibly because of the inflammatory consequences of western-style dietary habits, a major colon cancer risk factor. Machine learning methods designed to predict the cell-of-origin of cancer from patient-derived tumor samples confirmed that, in a substantial fraction of sporadic cases, the origins of colon cancer reside in secretory lineages and not in stem cells.

Global identification of new substrates for the yeast endoribonuclease, RNase mitochondrial RNA processing (MRP).

RNase mitochondrial RNA processing (MRP) is an essential, evolutionarily conserved endoribonuclease composed of 10 different protein subunits and a single RNA. RNase MRP has established roles in multiple pathways including ribosome biogenesis, cell cycle regulation, and mitochondrial DNA replication. Although each of these functions is important to cell growth, additional functions may exist given the essential nature of the complex. To identify novel RNase MRP substrates, we utilized RNA immunoprecipitation and microarray chip analysis to identify RNA that physically associates with RNase MRP. We identified several new potential substrates for RNase MRP including a cell cycle-regulated transcript, CTS1; the yeast homolog of the mammalian p27(Kip1), SIC1; and the U2 RNA component of the spliceosome. In addition, we found RNase MRP to be involved in the regulation of the Ty1 transposon RNA. These results reinforce and broaden the role of RNase MRP in cell cycle regulation and help to identify new roles of this endoribonuclease.

Occurrence of Meloidogyne fallax in North America, and Molecular Characterization of M. fallax and M. minor from U.S. Golf Course Greens.

Several species of root-knot nematodes (Meloidogyne spp.) are known to have significant presence on turfgrass in golf course greens, particularly in the western United States. Nematodes isolated from a golf course in King County, WA were identified as Meloidogyne minor based on analysis of the large ribosomal subunit (LSU 28S D2-D3 expansion segment), the internal transcribed spacers 1 and 2 (ITS rDNA), the intergenic spacer region 2 (IGS2), and the nuclear protein-coding gene Hsp90. Sequence-characterized amplified region (SCAR) primers that were originally designed to be specific for M. fallax were found to cross-react with M. minor. A population from California was determined to be M. fallax based on juvenile tail morphology and analysis of the ribosomal markers and Hsp90, comprising the first report of this species in North America. Using trees based on Hsp90 genomic alignments, the phylogenetic relationships of these populations and known root-knot nematode species were congruent with previous trees based on ribosomal genes. Resolution of M. fallax and M. chitwoodi using Hsp90 was equivalent to species separation obtained with 28S or 18S rDNA alignments. The strengths and weaknesses of ribosomal and Hsp90 markers, and the use of SCAR polymerase chain reaction as diagnostic tools are discussed.

Femoral Neck Fracture in a Pediatric Patient with Primary Hyperparathyroidism.

Case: A previously healthy 11-year-old girl underwent expedited surgical fixation of a femoral neck fracture sustained while jump-roping. After further work up, she was diagnosed with primary hyperparathyroidism. Parathyroidectomy of a hypertrophic adenoma proved curative. Now, five months post left hip surgery, the patient is pain-free and walks without a limp. Conclusion: We describe the first published case of primary hyperparathyroidism presenting as a pathologic hip fracture in a child. Although presentation with a fracture is exceedingly rare, bone pain is a frequent complaint of pediatric hyperparathyroidism. Orthopedic surgeons may find themselves the front-line caregivers for the condition.

Paneth cells as the origin of intestinal cancer in the context of inflammation.

Paneth cells (PCs), responsible for the secretion of antimicrobial peptides in the small intestine and for niche support to Lgr5+ crypt-base columnar stem cells (CBCs), have been shown to respond to inflammation by dedifferentiating into stem-like cells in order to sustain a regenerative response1,2. Therefore, PCs may represent the cells-of-origin of intestinal cancer in the context of inflammation. To test this hypothesis, we targeted Apc, Kras, and Tp53 mutations in Paneth cells by Cre-Lox technology and modelled inflammation by dextran sodium sulfate (DSS) administration. PC-specific loss of Apc resulted in multiple small intestinal tumors, whereas Kras or Tp53 mutations did not. Compound Apc and Kras mutations in PCs resulted in a striking increase in tumor multiplicity even in the absence of the inflammatory insult. By combining scRNAseq with lineage tracing to capture the conversion of PCs into bona fide tumor cells, we show that they progress through a "revival stem cell" (RSC) state characterized by high Clusterin (Clu) expression and Yap1 signaling, reminiscent of what has been previously observed upon irradiation of the mouse digestive tract3. Accordingly, comparison of PC- and Lgr5-derived murine intestinal tumors revealed differences related to Wnt signaling and inflammatory pathways which match the dichotomy of CBCs and injury-induced RSCs4 between human sporadic colon cancers and those arising in the context of inflammatory bowel diseases. Last, we show that western-style dietary habits, known to trigger a low-grade inflammation throughout the intestinal tract, underlie the analogous dedifferentiation of Paneth cells and their acquisition of stem-like features. Taken together, our results show that intestinal cancer arises in the context of inflammation through the dedifferentiation of committed secretory lineages such as Paneth cells and the activation of the revival stem cell state. As such, a true quiescent stem cell identity may be hidden in fully committed and postmitotic lineages which, upon inflammation, support the regenerative response by re-entering the cell cycle and dedifferentiating into RSCs. The chronic nature of the tissue insult in inflammatory bowel diseases and even in the context of western-style dietary habits is likely to result in the expansion of cell targets for tumor initiation and progression.

The Clinical Outcomes of Radiocapitellar Reconstruction With Radiocapitellar Arthroplasty: A Systematic Review and Meta-Analysis.

Background: Radiocapitellar arthroplasty fills a treatment void for young patients who experience isolated capitellar fractures or radiocapitellar osteoarthritis who are not candidates for total elbow arthroplasty. The outcomes of this procedure are sparsely reported. We designed a meta-analysis to determine the utility of radiocapitellar arthroplasty with respect to functional and patient reported outcomes. Methods: The PubMed database was searched for relevant studies. Only studies published in English language that assessed patient reported outcomes following radiocapitellar arthroplasty were included in this study. Reporting followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses for 2020. Results: The initial review yielded 562 studies that met the criteria. After excluding duplications and confounding factors, eight case series were identified for review. Of the eight studies, seven were eligible for inclusion in the meta-analysis for Mayo Elbow Performance Score, flexion-extension arc, and pronation-supination arc. The pooled standard mean difference was found to be statistically significant between pre-operative and post-operative outcomes for Mayo Elbow Performance Score (SMD = 3.04, 95% CI [2.40, 3.67]), flexion-extension arc (SMD = 1.28, 95% CI [0.73, 1.83]), and pronation-supination arc (SMD = 0.81, 95% CI [0.43, 1.18]). Cochran's Q-test and I2 statistics indicated statistically significant heterogeneity for Mayo Elbow Performance Score (p = .04, I2 = 54%) and flexion-extension arc (p < .01, I2 = 67%). Conclusions: Patients undergoing radiocapitellar arthroplasty showed statistically significant improvements in flexion-extension arc, pronation-supination arc, and Mayo Elbow Performance Scores compared to pre-operative measures.

Osteocutaneous Radial Forearm Flap: Harvest Technique and Prophylactic Volar Locked Plating.

Background: The osteocutaneous radial forearm (OCRF) flap is a variation of the traditional radial forearm flap with incorporation of an anterolateral segment of corticocancellous bone of the radius, periosteum, and overlying skin. The OCRF flap is indicated in traumatic injuries or extirpation defects with segmental bone loss and is well suited to foot and ankle reconstruction due to its thin pliable skin. Methods: In this single-center case series, a retrospective review was conducted to identify patients who underwent OCRF free flap for foot and ankle reconstruction that required harvest of more than 50% of the cross-sectional area of the radius with prophylactic volar locked plating of the donor site. Outcome measures included flap failure rates, postoperative fracture, thrombotic events, time to follow-up, and time to full weightbearing. Flap harvest technique is extensively discussed. Results: Six cases were included in this series. There were no flap failures or thrombotic events. Recipient site healing was confirmed in all patients, with partial distal skin paddle loss in one patient requiring operative debridement. No patients sustained donor site complications or functional impairment. Full lower extremity weightbearing was achieved at 12.4 ± 3.3 weeks after surgery. Conclusions: The OCRF free flap transfer provides a reliable means of obtaining thin, supple soft tissue coverage with a large, vascularized segment of bone for reconstruction in the foot and ankle. Here, we describe use of more than 50% of the cross-sectional area of the radius with volar locked prophylactic plating. These updates expand use of this reconstructive technique.

The origin of intestinal cancer in the context of inflammation.

According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, was shown to suppress intestinal stemness. Here, we employed Paneth cells (PCs) as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation. Upon inflammation, PC-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in inflammatory bowel disease (IBD) patients but also of a larger fraction of sporadic colon cancers. The latter is likely due to the inflammatory consequences of Western-style dietary habits, the major colon cancer risk factor. Computational methods designed to predict the cell-of-origin of cancer confirmed that, in a substantial fraction of sporadic colon cancers the cells-of-origin are secretory lineages and not stem cells.

Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components.

RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.

Role of RNase MRP in viral RNA degradation and RNA recombination.

RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

Root-Knot Nematodes in Golf Course Greens of the Western United States.

A survey of 238 golf courses in 10 states of the western United States found root-knot nematodes (Meloidogyne spp.) in 60% of the putting greens sampled. Sequence and phylogenetic analyses of 18S rRNA, D2-D3 of 28S rRNA, internal transcribed spacer-rRNA, and mitochondrial DNA gene sequences were used to identify specimens from 110 golf courses. The most common species, Meloidogyne naasi, was found in 58 golf courses distributed from Southern California to Washington in the coastal or cooler areas of those states. In the warmer regions of the Southwest, M. marylandi was recovered from 38 golf courses and M. graminis from 11 golf courses. This constitutes the first report of M. marylandi in Arizona, California, Hawaii, Nevada, and Utah, and the first report of M. graminis in Arizona, Hawaii, and Nevada. Two golf courses in Washington were infested with M. minor, the first record of this nematode in the Western Hemisphere. Columbia root-knot nematode, M. chitwoodi, was found in a single golf course in California. Polymerase chain reaction restriction fragment length polymorphism of the intergenic region between the cytochrome oxidase and 16S rRNA genes in the mitochondrial genome with restriction enzyme SspI was able to distinguish populations of M. graminis from M. marylandi, providing a fast and inexpensive method for future diagnosis of these nematodes from turf.

KMT9 Controls Stemness and Growth of Colorectal Cancer.

Colorectal cancer is among the leading causes of cancer-associated deaths worldwide. Treatment failure and tumor recurrence due to survival of therapy-resistant cancer stem/initiating cells represent major clinical issues to overcome. In this study, we identified lysine methyltransferase 9 (KMT9), an obligate heterodimer composed of KMT9α and KMT9ß that monomethylates histone H4 at lysine 12 (H4K12me1), as an important regulator in colorectal tumorigenesis. KMT9α and KMT9ß were overexpressed in colorectal cancer and colocalized with H4K12me1 at promoters of target genes involved in the regulation of proliferation. Ablation of KMT9α drastically reduced colorectal tumorigenesis in mice and prevented the growth of murine as well as human patient-derived tumor organoids. Moreover, loss of KMT9α impaired the maintenance and function of colorectal cancer stem/initiating cells and induced apoptosis specifically in this cellular compartment. Together, these data suggest that KMT9 is an important regulator of colorectal carcinogenesis, identifying KMT9 as a promising therapeutic target for the treatment of colorectal cancer. SIGNIFICANCE: The H4K12 methyltransferase KMT9 regulates tumor cell proliferation and stemness in colorectal cancer, indicating that targeting KMT9 could be a useful approach for preventing and treating this disease.

Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop.

Enhancement of Wnt signaling is fundamental for stem cell function during intestinal regeneration. Molecular modules control Wnt activity by regulating signal transduction. CD44 is such a positive regulator and a Wnt target gene. While highly expressed in intestinal crypts and used as a stem cell marker, its role during intestinal homeostasis and regeneration remains unknown. Here we propose a CD44 positive-feedback loop that boosts Wnt signal transduction, thus impacting intestinal regeneration. Excision of Cd44 in Cd44fl/fl;VillinCreERT2 mice reduced Wnt target gene expression in intestinal crypts and affected stem cell functionality in organoids. Although the integrity of the intestinal epithelium was conserved in mice lacking CD44, they were hypersensitive to dextran sulfate sodium, and showed more severe inflammation and delayed regeneration. We localized the molecular function of CD44 at the Wnt signalosome, and identified novel DVL/CD44 and AXIN/CD44 complexes. CD44 thus promotes optimal Wnt signaling during intestinal regeneration.

A specialized processing body that is temporally and asymmetrically regulated during the cell cycle in Saccharomyces cerevisiae.

RNase mitochondrial RNA processing (MRP) is an essential ribonucleoprotein endoribonuclease that functions in the degradation of specific mRNAs involved in cell cycle regulation. We have investigated where this processing event occurs and how it is regulated. As expected, results demonstrate that RNase MRP is predominantly localized in the nucleolus, where it processes ribosomal RNAs. However, after the initiation of mitosis, RNase MRP localizes throughout the entire nucleus and in a single discrete cytoplasmic spot that persists until the completion of telophase. Furthermore, this spot was asymmetrically found in daughter cells, where the RNase MRP substrate, CLB2 mRNA, localizes. Both the mitotic exit network and fourteen early anaphase release pathways are nonessential but important for the temporal changes in localization. Asymmetric localization was found to be dependent on the locasome. The evidence suggests that these spots are specialized processing bodies for the degradation of transcripts that are cell cycle regulated and daughter cell localized. We have called these TAM bodies for temporal asymmetric MRP bodies.

The inflammatory pathogenesis of colorectal cancer.

The mutational landscape of colorectal cancer (CRC) does not enable predictions to be made about the survival of patients or their response to therapy. Instead, studying the polarization and activation profiles of immune cells and stromal cells in the tumour microenvironment has been shown to be more informative, thus making CRC a prototypical example of the importance of an inflammatory microenvironment for tumorigenesis. Here, we review our current understanding of how colon cancer cells interact with their microenvironment, comprised of immune cells, stromal cells and the intestinal microbiome, to suppress or escape immune responses and how inflammatory processes shape the immune pathogenesis of CRC.