Implementation science in humanitarian assistance: applying a novel approach for humanitarian care optimization.

Humanitarian assistance is hindered by a lack of strategies to optimize care delivery through research and organized networks. Distinct from global health, humanitarian assistance struggles to address its multifaceted challenges, including duplicative resources, uncoordinated communication, unregulated staff expertise and safety, financial waste, and poor-quality metrics and care delivery. Implementation science provides an exciting and underutilized approach that can be applied to address these challenges, by studying how to effectively design, implement, optimize, and scale evidence-based interventions. Though successful in well-resourced and global health systems, implementation science approaches are rare in humanitarian assistance. Adopting implementation science approaches including identifying determinants, creating accessible evidence-based intervention bundles, adapting study methodologies for the humanitarian context, and partnering with implementation experts could make these promising approaches more accessible for thousands of humanitarian actors delivering healthcare for millions of vulnerable patients worldwide.

Understanding pediatric surgical needs in North Korea: a modeling analysis.

Background: Sixty-five percent of children worldwide lack access to surgical care, the majority of whom live in low-income and middle-income countries (LMICs). Developing surgical infrastructure requires information on surgical need; however, this information is often limited in LMICs. North Korea (Democratic People's Republic of Korea, DPRK) has a low amount of publicly available data. Here, we analyzed available modeled data to understand the causes of pediatric deaths due to conditions treatable with surgery in DPRK. Methods: We used World Bank data and models from the Institute for Health Metrics and Evaluation to identify causes of pediatric deaths affecting pediatric patients (age <20 years). We compared mortality of disease between DPRK and countries with similar economic status. Results: Between 1990 and 2019, the number of overall pediatric deaths has decreased. In 2019, 32.2% of all pediatric deaths in DPRK were caused by surgical conditions. The leading categories of surgical conditions were injuries (53.9%), congenital conditions (34.2%), tumors (8.8%), and abdominal conditions (3.2%). DPRK has a lower relative rate of pediatric deaths compared with other LMICs with similar gross domestic product per capita. However, it has a higher relative rate of pediatric deaths due to conditions requiring treatment with surgery. Transport injuries contribute significantly to the high rate of pediatric deaths in DPRK. Conclusions: Although DPRK may be allocating overall resources toward pediatric healthcare more efficiently than economic peers, DPRK may benefit from improvement in pediatric surgical capacity. Improved availability of data and close international collaboration could be potential solutions to bridge this gap.

Epitranscriptomic Reader YTHDF2 Regulates SEK1( MAP2K4 )-JNK-cJUN Inflammatory Signaling in Astrocytes during Neurotoxic Stress.

As the most abundant glial cells in the CNS, astrocytes dynamically respond to neurotoxic stress, however, the key molecular regulators controlling the inflammatory status of these sentinels during neurotoxic stress have remained elusive. Herein, we demonstrate that the m6A epitranscriptomic mRNA modification tightly regulates the pro-inflammatory functions of astrocytes. Specifically, the astrocytic neurotoxic stresser, manganese (Mn), downregulated the m6A reader YTHDF2 in human and mouse astrocyte cultures and in the mouse brain. Functionally, YTHDF2 knockdown augmented, while its overexpression dampened, neurotoxic stress induced proinflammatory response, suggesting YTHDF2 serves as a key upstream regulator of inflammatory responses in astrocytes. Mechnistically, YTHDF2 RIP-sequencing identified MAP2K4 ( MKK4; SEK1) mRNA as a YTHDF2 target influencing inflammatory signaling. Our target validation revealed Mn-exposed astrocytes mediates proinflammatory response by activating the phosphorylation of SEK1, JNK, and cJUN signaling. Collectively, YTHDF2 serves a key upstream 'molecular switch' controlling SEK1( MAP2K4 )-JNK-cJUN proinflammatory signaling in astrocytes.

Out-of-pocket costs and catastrophic healthcare expenditure for families of children requiring surgery in sub-Saharan Africa.

BACKGROUND: Out-of-pocket healthcare costs leading to catastrophic healthcare expenditure pose a financial threat for families of children undergoing surgery in Sub-Saharan African countries, where universal healthcare coverage is often insufficient. METHODS: A prospective clinical and socioeconomic data collection tool was used in African hospitals with dedicated pediatric operating rooms installed philanthropically. Clinical data were collected via chart review and socioeconomic data from families. The primary indicator of economic burden was the proportion of families with catastrophic healthcare expenditures. Secondary indicators included the percentage who borrowed money, sold possessions, forfeited wages, and lost a job secondary to their child's surgery. Descriptive statistics and multivariate logistic regression were performed to identify predictors of catastrophic healthcare expenditure. RESULTS: In all, 2,296 families of pediatric surgical patients from 6 countries were included. The median annual income was $1,000 (interquartile range 308-2,563), whereas the median out-of-pocket cost was $60 (interquartile range 26-174). Overall, 39.9% (n = 915) families incurred catastrophic healthcare expenditure, 23.3% (n = 533) borrowed money, 3.8% (n = 88%) sold possessions, 26.4% (n = 604) forfeited wages, and 2.3% (n = 52) lost a job because of the child's surgery. Catastrophic healthcare expenditure was associated with older age, emergency cases, need for transfusion, reoperation, antibiotics, and longer length of stay, whereas the subgroup analysis found insurance to be protective (odds ratio 0.22, P = .002). CONCLUSION: A full 40% of families of children in sub-Saharan Africa who undergo surgery incur catastrophic healthcare expenditure, shouldering economic consequences such as forfeited wages and debt. Intensive resource utilization and reduced insurance coverage in older children may contribute to a higher likelihood of catastrophic healthcare expenditure and can be insurance targets for policymakers.

Surgeon behaviors related to engaging patients in smoking cessation at the time of elective surgery.

BACKGROUND: Despite the abundance of evidence supporting smoking cessation before elective surgery, there is wide variation in surgeon adherence to these best practices. METHODS: This qualitative study used convenience sampling to recruit General Surgery trained surgeons. Surgeons participated in semi-structured interviews based on domains from the Theoretical Domains Framework (TDF). Content analysis was guided by the TDF. RESULTS: Of the 14 TDF domains, social or professional role/identity, memory, attention and decision processes, environmental context and resources, and beliefs about consequences emerged most frequently. Mapping these domains to the Behavior Change Wheel identified education, enablement, and incentivization as effective intervention functions. CONCLUSIONS: Using the TDF, this study identified a widespread sense of responsibility among surgeons to engage patients in perioperative smoking cessation despite workplace barriers and lacking resources. These findings provide valuable insight to facilitate surgeon participation in health promotion through targeted, theory-based interventions informed by surgeon identified barriers to perioperative smoking cessation.

Bile leak incidence, risk factors and associated outcomes in patients undergoing hepatectomy: a contemporary NSQIP propensity matched analysis.

BACKGROUND: Despite advances in surgical technique, bile leak remains a common complication following hepatectomy. We sought to identify incidence of, risk factors for, and outcomes associated with biliary leak. STUDY DESIGN: This is an ACS-NSQIP study. Distribution of bile leak stratified by surgical approach and hepatectomy type were identified. Univariate and multivariate factors associated with bile leak and outcomes were evaluated. RESULTS: Robotic hepatectomy was associated with less bile leak (5.4% vs. 11.4%; p < 0.001) compared to open. There were no significant differences in bile leak between robotic and laparoscopic hepatectomy (5.4% vs. 5.3%; p = 0.905, respectively). Operative factors risk factors for bile leak in patients undergoing robotic hepatectomy included right hepatectomy [OR 4.42 (95% CI 1.74-11.20); p = 0.002], conversion [OR 4.40 (95% CI 1.39-11.72); p = 0.010], pringle maneuver [OR 3.19 (95% CI 1.03-9.88); p = 0.044], and drain placement [OR 28.25 (95% CI 8.34-95.72); p < 0.001]. Bile leak was associated with increased reoperation (8.7% vs 1.7%, p < 0.001), 30-day readmission (26.6% vs 6.8%, p < 0.001), 30-day mortality (2% vs 0.9%, p < 0.001), and complications (67.2% vs 23.4%, p < 0.001) for patients undergoing MIS hepatectomy. CONCLUSION: While MIS confers less risk for bile leak than open hepatectomy, risk factors for bile leak in patients undergoing MIS hepatectomy were identified. Bile leaks were associated with multiple additional complications, and the robotic approach had an equal risk for bile leak than laparoscopic in this time period.

Utility of Perioperative Measurement of Cell-Free DNA and Circulating Tumor DNA in Informing the Prognosis of GI Cancers: A Systematic Review.

PURPOSE: Current surveillance imaging and tumor markers lack sensitivity for the early detection of recurrence in GI cancers. This study critically evaluates the current literature on the role of sequential measurement of circulating tumor DNA (ctDNA) before and after curative resection in informing recurrence. METHODS: A systematic search using a predefined, registered protocol was conducted for studies published between January 2010 and May 2020. Included studies described patients with GI cancers treated with curative-intent surgical resection and measurement of ctDNA both before and after surgery. Patients were divided into three groups on the basis of the presence or absence of ctDNA at these time points. The primary outcome was recurrence-free survival (RFS). RESULTS: The search yielded 3,873 articles; five met the inclusion criteria and collectively evaluated 57 patients. Pooled median RFS was 62 months (interquartile range 19 to not reached). Although median RFS was not reached in group 1 (- to -) or group 2 (+ to -), median RFS in group 3 (+ to +) was 15 months (interquartile range 9.6-60.4 months). Cox hazard ratio was 4.46 (95% CI, 1.17 to 16.99; P = .028) between group 1 and group 2, and 10.47 (95% CI, 2.91 to 37.74; P < .001) between group 2 and group 3. CONCLUSION: Detectable ctDNA, either preoperatively or postoperatively, and its persistence after curative surgery are associated with a greater risk of recurrence and decreased RFS in GI cancers. Thus, perioperative measurement of ctDNA may be a useful postoperative risk stratification tool and guide additional therapies.

Novel response to neoadjuvant anti-PD1 therapy for a patient with retrocaval melanotic schwannoma.

Melanotic schwannoma is a rare nerve sheath tumor composed of melanin-producing Schwann cells with the potential for metastasis. These tumors can be associated with familial tumor syndromes and can cause significant symptoms related to nerve compression and mass effect. Due to the rarity of these lesions, they can be initially misidentified as melanocytomas, pigmented dermatofibrosarcoma protuberans, neurofibromas or malignant melanomas. Surgical excision is the mainstay of treatment with limited benefit from adjuvant systemic chemotherapy or radiation. Modern treatments with immune checkpoint blockade have demonstrated significant improvements in progression-free and overall survival for a variety of cancer histologies; however, anti-PD1 therapy has yet to be evaluated in patients with melanotic schwannoma. This report demonstrates a significant improvement in symptomatology and tumor stability with neoadjuvant anti-PD1 therapy for a retrocaval melanotic schwannoma initially masquerading as malignant melanoma. This report demonstrates the potential benefit of a novel therapeutic option for patients with melanotic schwannoma.

N6-Adenosine Methylation of Socs1 mRNA Is Required to Sustain the Negative Feedback Control of Macrophage Activation.

Bacterial infection triggers a cytokine storm that needs to be resolved to maintain the host's wellbeing. Here, we report that ablation of m6A methyltransferase subunit METTL14 in myeloid cells exacerbates macrophage responses to acute bacterial infection in mice, leading to high mortality due to sustained production of pro-inflammatory cytokines. METTL14 depletion blunts Socs1 m6A methylation and reduces YTHDF1 binding to the m6A sites, which diminishes SOCS1 induction leading to the overactivation of TLR4/NF-κB signaling. Forced expression of SOCS1 in macrophages depleted of METTL14 or YTHDF1 rescues the hyper-responsive phenotype of these macrophages in vitro and in vivo. We further show that LPS treatment induces Socs1 m6A methylation and sustains SOCS1 induction by promoting Fto mRNA degradation, and forced FTO expression in macrophages mimics the phenotype of METTL14-depleted macrophages. We conclude that m6A methylation-mediated SOCS1 induction is required to maintain the negative feedback control of macrophage activation in response to bacterial infection.

Functional role of Tet-mediated RNA hydroxymethylcytosine in mouse ES cells and during differentiation.

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.

Control of Early B Cell Development by the RNA N6-Methyladenosine Methylation.

The RNA N6-methyladenosine (m6A) methylation is installed by the METTL3-METTL14 methyltransferase complex. This modification has critical regulatory roles in various biological processes. Here, we report that deletion of Mettl14 dramatically reduces mRNA m6A methylation in developing B cells and severely blocks B cell development in mice. Deletion of Mettl14 impairs interleukin-7 (IL-7)-induced pro-B cell proliferation and the large-pre-B-to-small-pre-B transition and causes dramatic abnormalities in gene expression programs important for B cell development. Suppression of a group of transcripts by cytoplasmic m6A reader YTHDF2 is critical to the IL-7-induced pro-B cell proliferation. In contrast, the block in the large-pre-B-to-small-pre-B transition is independent of YTHDF1 or YTHDF2 but is associated with a failure to properly upregulate key transcription factors regulating this transition. Our data highlight the important regulatory roles of the RNA m6A methylation and its reader proteins in early B cell development.

Molecular biology of oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OCSCC) is a heterogeneous and complex disease that arises due to dysfunction of multiple molecular signaling pathways. Recent advances in high-throughput genetic sequencing technologies coupled with innovative analytical techniques have begun to characterize the molecular determinants driving OCSCC. An understanding of the key molecular signaling networks underlying the initiation and progression of is essential for informing treatment of the disease. In this chapter, we discuss recent findings of key genes altered in OCSCC and potential treatments targeting these genes.

Robotic Distal Pancreatectomy and Splenectomy for an Intrapancreatic Hepatocellular Carcinoma: A Case Report and Review of the Literature.

Background: Liver parenchyma that resides outside of the normal hepatic confines is defined as accessory liver if in communication with the native biliary tree, or ectopic liver (EL) if it is not. EL can develop in a variety of tissues, including but not limited to the gallbladder, the hepatic ligaments, the pancreas, and retroperitoneum. EL has an increased propensity for malignant degeneration resulting in hepatocellular carcinoma (HCC). Presentation: A 67-year-old Korean male presented with epigastric discomfort and was found to have an elevation in his transaminases. Cross-sectional imaging demonstrated a 1.3 cm solid mass in the body of the pancreas with features concerning for either a pancreatic ductal adenocarcinoma or pancreatic neuroendocrine tumor. Subsequent endoscopic ultrasound and fine needle aspiration demonstrated cells of epithelial origin with hepatocellular differentiation. A robotic-assisted distal pancreatectomy and splenectomy was performed with final pathology demonstrating a well-differentiated HCC. Conclusions: EL with malignant degeneration resulting in HCC requires surgical excision. The majority of patients reported with EL resulting in HCC in the pancreas have had the tumors located in the body and tail. Therefore, definitive treatment requires distal pancreatectomy and splenectomy. Herein, we describe the presentation, workup, and definitive treatment of HCC arising in the pancreas.

The RNA-binding protein FMRP facilitates the nuclear export of N6-methyladenosine-containing mRNAs.

N6-Methyladenosine (m6A) is the most abundant post-transcriptional mRNA modification in eukaryotes and exerts many of its effects on gene expression through reader proteins that bind specifically to m6A-containing transcripts. Fragile X mental retardation protein (FMRP), an RNA-binding protein, has previously been shown to affect the translation of target mRNAs and trafficking of mRNA granules. Loss of function of FMRP causes fragile X syndrome, the most common form of inherited intellectual disability in humans. Using HEK293T cells, siRNA-mediated gene knockdown, cytoplasmic and nuclear fractions, RNA-Seq, and LC-MS/MS analyses, we demonstrate here that FMRP binds directly to a collection of m6A sites on mRNAs. FMRP depletion increased mRNA m6A levels in the nucleus. Moreover, the abundance of FMRP targets in the cytoplasm relative to the nucleus was decreased in Fmr1-KO mice, an effect also observed in highly methylated genes. We conclude that FMRP may affect the nuclear export of m6A-modified RNA targets.

Single base resolution mapping of 2'-O-methylation sites in human mRNA and in 3' terminal ends of small RNAs.

The post-transcriptional modification 2'-O-Methyl (2'OMe) could be present on the ribose of all four ribonucleosides, and is highly prevalent in a wide variety of RNA species, including the 5' RNA cap of viruses and higher eukaryotes, as well as internally in transfer RNA and ribosomal RNA. Recent studies have suggested that 2'OMe is also located internally in low-abundance RNA species such as viral RNA and mRNA. To profile 2'OMe on different RNA species, we have developed Nm-seq, which could identify 2'OMe sites at single base resolution. Nm-seq is particularly useful for identifying 2'OMe sites located at the 3' terminal ends of small RNAs. Here, we present an optimized protocol for Nm-seq and a protocol for applying Nm-seq to identify 2'OMe sites on small RNA 3' terminal ends.

High-Resolution Mapping of N 6-Methyladenosine Using m6A Crosslinking Immunoprecipitation Sequencing (m6A-CLIP-Seq).

N 6-Methyladenosine, an abundant chemical modification in mRNA, plays crucial roles in regulating gene expression and biological processes. Research on m6A and its functions has progressed rapidly in the past few years, aided substantially by advances in high-throughput sequencing-based methods to profile m6A along the transcriptome. We present here a protocol for m6A crosslinking immunoprecipitation sequencing (m6A-CLIP-seq), which profiles m6A on mRNA at high resolution from as little as 1 µg of poly(A)-selected mRNA.

Dynamic transcriptomic m6A decoration: writers, erasers, readers and functions in RNA metabolism.

N6-methyladenosine (m6A) is a chemical modification present in multiple RNA species, being most abundant in mRNAs. Studies on enzymes or factors that catalyze, recognize, and remove m6A have revealed its comprehensive roles in almost every aspect of mRNA metabolism, as well as in a variety of physiological processes. This review describes the current understanding of the m6A modification, particularly the functions of its writers, erasers, readers in RNA metabolism, with an emphasis on its role in regulating the isoform dosage of mRNAs.

Identifying the m6A Methylome by Affinity Purification and Sequencing.

N 6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic mRNA, and is newly emerging as a key posttranscriptional mRNA regulator. Recent research has uncovered insight into the location and function of m6A sites on a large scale, in part due to the transcriptome-wide identification of m6A sites by high-throughput sequencing (m6A-seq). Here, we present a protocol for m6A-seq, which maps the m6A methylome by affinity purification and sequencing.

Epitranscriptomic influences on development and disease.

RNA contains over 150 types of chemical modifications. Although many of these chemical modifications were discovered several decades ago, their functions were not immediately apparent. Discoveries of RNA demethylases, along with advances in mass spectrometry and high-throughput sequencing techniques, have caused research into RNA modifications to progress at an accelerated rate. Post-transcriptional RNA modifications make up an epitranscriptome that extensively regulates gene expression and biological processes. Here, we present an overview of recent advances in the field that are shaping our understanding of chemical modifications, their impact on development and disease, and the dynamic mechanisms through which they regulate gene expression.