Complications and adverse events in lymphadenectomy of the inguinal area: worldwide expert consensus.

BACKGROUND: Inguinal lymph node dissection plays an important role in the management of melanoma, penile and vulval cancer. Inguinal lymph node dissection is associated with various intraoperative and postoperative complications with significant heterogeneity in classification and reporting. This lack of standardization challenges efforts to study and report inguinal lymph node dissection outcomes. The aim of this study was to devise a system to standardize the classification and reporting of inguinal lymph node dissection perioperative complications by creating a worldwide collaborative, the complications and adverse events in lymphadenectomy of the inguinal area (CALI) group. METHODS: A modified 3-round Delphi consensus approach surveyed a worldwide group of experts in inguinal lymph node dissection for melanoma, penile and vulval cancer. The group of experts included general surgeons, urologists and oncologists (gynaecological and surgical). The survey assessed expert agreement on inguinal lymph node dissection perioperative complications. Panel interrater agreement and consistency were assessed as the overall percentage agreement and Cronbach's α. RESULTS: Forty-seven experienced consultants were enrolled: 26 (55.3%) urologists, 11 (23.4%) surgical oncologists, 6 (12.8%) general surgeons and 4 (8.5%) gynaecology oncologists. Based on their expertise, 31 (66%), 10 (21.3%) and 22 (46.8%) of the participants treat penile cancer, vulval cancer and melanoma using inguinal lymph node dissection respectively; 89.4% (42 of 47) agreed with the definitions and inclusion as part of the inguinal lymph node dissection intraoperative complication group, while 93.6% (44 of 47) agreed that postoperative complications should be subclassified into five macrocategories. Unanimous agreement (100%, 37 of 37) was achieved with the final standardized classification system for reporting inguinal lymph node dissection complications in melanoma, vulval cancer and penile cancer. CONCLUSION: The complications and adverse events in lymphadenectomy of the inguinal area classification system has been developed as a tool to standardize the assessment and reporting of complications during inguinal lymph node dissection for the treatment of melanoma, vulval and penile cancer.

Ancestry-associated co-alteration landscape of KRAS and EGFR-altered non-squamous NSCLC.

Racial/ethnic disparities mar NSCLC care and treatment outcomes. While socioeconomic factors and access to healthcare are important drivers of NSCLC disparities, a deeper understanding of genetic ancestry-associated genomic landscapes can better inform the biology and the treatment actionability for these tumors. We present a comprehensive ancestry-based prevalence and co-alteration landscape of genomic alterations and immunotherapy-associated biomarkers in patients with KRAS and EGFR-altered non-squamous (non-Sq) NSCLC. KRAS was the most frequently altered oncogene in European (EUR) and African (AFR), while EGFR alterations predominated in East Asian (EAS), South Asian (SAS), and Admixed American (AMR) groups, consistent with prior studies. As expected, STK11 and KEAP1 alterations co-occurred with KRAS alterations while showing mutual exclusivity with EGFR alterations. EAS and AMR KRAS-altered non-Sq NSCLC showed lower rates of co-occurring STK11 and KEAP1 alterations relative to other ancestry groups. Ancestry-specific co-alterations included the co-occurrence of KRAS and GNAS alterations in AMR, KRAS, and ARID1A alterations in SAS, and the mutual exclusivity of KRAS and NF1 alterations in the EUR and AFR ancestries. Contrastingly, EGFR-altered tumors exhibited a more conserved co-alteration landscape across ancestries. AFR exhibited the highest tumor mutational burden, with potential therapeutic implications for these tumors.

Malnutrition in emergency general surgery: a survey of National Emergency Laparotomy Audit Leads.

BACKGROUND: Patients who are malnourished and have emergency general surgery, such as a laparotomy, have worse outcomes than those who are not malnourished. It is paramount to identify these patients and minimise this risk. This study aimed to describe current practices in identifying malnutrition in patients undergoing a laparotomy, specifically focusing on screening, assessment, nutrition pathways and barriers encountered by clinicians. METHODS: Following piloting and validity assessment, anaesthetic and surgical National Emergency Laparotomy Audit (NELA) Leads at hospitals across England and Wales were emailed an invitation to a survey. Responses were gathered using Qualtrics. Descriptive analysis and correlation with laparotomy volume and professional role were performed in SPSSv26. University of Sheffield ethical approval was obtained (UREC 046205). The results from the survey are reported according to the CHERRIES guidelines. RESULTS: The survey was completed by 166/289 NELA Leads from 117/167 hospitals (57.4% and 70.1% response rates, respectively). Participants reported low rates of nutritional screening (42/166; 25.3%) and assessment (26/166; 15.7%) for malnutrition preoperatively. More than one third of respondents (40.1%) had no awareness of local screening tools; indeed, the Malnutrition Universal Screening Tool (MUST) was used by approximately half of respondents (56.6%). Contrary to guidelines, NELA Leads report albumin levels continue to be used to determine malnutrition risk (73.5%; 122/166). Postoperative nutrition pathways were common (71.7%; 119/166). Reported barriers to nutritional screening and assessment included a lack of time, training and education, organisational support and ownership. Participants indicated nutrition risk is inadequately identified and is an important missing data item from NELA. There was no significant correlation with hospital laparotomy volume in relation to screening or assessment for malnutrition, the use of nutritional support pathways or organisational barriers. There was interprofessional agreement across a number of domains, although some differences did exist. CONCLUSIONS: Wide variation exists in the current practice of identifying malnutrition risk in NELA patients. Barriers include a lack of time, knowledge and ownership. Nutrition pathways that encompass the preoperative phase and incorporation of nutrition data in NELA may support improvements in care.

Hospital outbreak of Carbapenem-resistant acinetobacter baumannii in the context of local facility transmission.

Carbapenem-resistant Acinetobacter baumannii are of increasing concern in the health care setting. We describe a cluster of 9 cases in hospitalized patients over a 3-month period that reflected ongoing community transmission from high-risk facilities. Robust surveillance and knowledge of local epidemiology are critical to mitigating onward transmission in the health care setting.

Diagnostic miRNA Signatures in Paired Tumor, Plasma, and Urine Specimens From Renal Cell Carcinoma Patients.

BACKGROUND: The incidence of patients diagnosed with renal cell carcinoma (RCC) is increasing. There are no approved biofluid biomarkers for routine diagnosis of RCC patients. This retrospective study aims to identify cell-free microRNA (cfmiR) signatures in urine samples that can be utilized as biomarkers for early diagnosis of sporadic RCC patients. METHODS: Tissue, plasma, and urine samples (n = 221) from 56 sporadic RCC patients and respective normal healthy donors were profiled for 2083 microRNAs (miRs) using the next-generation sequencing-based HTG EdgeSeq miR Whole Transcriptome Assay. DESeq2 (FC |1.2|, false discovery rate <0.05) was performed to identify differentially expressed miRs. Data from RCC tissue samples of The Cancer Genome Atlas database were used for miR validation. RESULTS: We found a 10-miR signature that distinguished RCC tissues from remote normal kidney tissue or benign kidney lesion samples. Additionally, we identified subtype-specific miRs (miR-122-5p, miR-210-3p, and miR-21-3p) and miRs specific for all RCC subtypes (miR-106b-3p, miR-629-5p, and miR-885-5p). We observed that miR-155-5p was associated with tumor size. Using The Cancer Genome Atlas data sets, we validated the miRs found in RCC tissue samples. In plasma or urine analysis, we found cfmiRs that were consistently and significantly upregulated in RCC tissue samples. A 15-cfmiR signature was proposed in urine samples of RCC patients, of which miR-1275 was consistently upregulated in tissue, plasma, and urine samples. CONCLUSIONS: This integrative study found diagnostic miRs/cfmiRs for RCC patients, which were validated using The Cancer Genome Atlas data sets. Distinctive cfmiR signatures found in urine may have clinical utility for the diagnosis of RCC.

Leishmania genetic exchange is mediated by IgM natural antibodies.

Host factors that mediate Leishmania genetic exchange are not well defined. Here we demonstrate that natural IgM (IgMn)1-4 antibodies mediate parasite genetic exchange by inducing the transient formation of a spherical parasite clump that promotes parasite fusion and hybrid formation. We establish that IgMn from Leishmania-free animals binds to the surface of Leishmania parasites to induce significant changes in the expression of parasite transcripts and proteins. Leishmania binding to IgMn is partially lost after glycosidase treatment, although parasite surface phosphoglycans, including lipophosphoglycan, are not required for IgMn-induced parasite clumping. Notably, the transient formation of parasite clumps is essential for Leishmania hybridization in vitro. In vivo, we observed a 12-fold increase in hybrid formation in sand flies provided a second blood meal containing IgMn compared with controls. Furthermore, the generation of recombinant progeny from mating hybrids and parental lines were only observed in sand flies provided with IgMn. Both in vitro and in vivo IgM-induced Leishmania crosses resulted in full genome hybrids that show equal patterns of biparental contribution. Leishmania co-option of a host natural antibody to facilitate mating in the insect vector establishes a new paradigm of parasite-host-vector interdependence that contributes to parasite diversity and fitness by promoting genetic exchange.

Identifying malnutrition in emergency general surgery: systematic review.

BACKGROUND: Emergency general surgery practice is high risk. Surgery is a key part of treatment, with resultant catabolic stress and frequent need for nutritional support. The aim of this study was to examine the current methods of defining and determining malnutrition in emergency general surgery. This included examining the use of nutrition screening and assessment tools and other measures of malnutrition. METHODS: MEDLINE, Embase, Cumulative Index to Nursing and Allied Health Literature, trial registries, and relevant journals published between January 2000 and January 2022 were searched for studies of adult patients with any emergency general surgery diagnosis, managed conservatively or operatively, with an assessment of nutritional status. Mixed populations were included if more than 50 per cent of patients were emergency general surgery patients or emergency general surgery results could be separately extracted. Studies in which patients had received nutritional support were excluded. The protocol was registered with PROSPERO, the international prospective register of systematic reviews (CRD42021285897). RESULTS: From 6700 studies screened, 324 full texts were retrieved and 31 were included in the analysis. A definition of malnutrition was provided in 23 studies (75 per cent), with nutritional status being determined by a variety of methods. A total of seven nutrition screening tools and a total of nine 'assessment' tools were reported. To define malnutrition, the most commonly used primary or secondary marker of nutritional status was BMI, followed by albumin level. CONCLUSION: Wide variation exists in approaches to identify malnutrition risk in emergency general surgery patients, using a range of tools and nutrition markers. Future studies should seek to standardize nutrition screening and assessment in the emergency general surgery setting as two discrete processes. This will permit better understanding of malnutrition risk in surgical patients.

Zoledronic Acid Prevents Bone Resorption Caused by the Combination of Radium-223, Abiraterone Acetate, and Prednisone in an Intratibial Prostate Cancer Mouse Model.

An increased risk of non-pathological fractures in patients with prostate cancer and bone metastases has been associated with combination treatment with radium-223, abiraterone, and prednisone/prednisolone in the absence of bone-protecting agents. Here, we investigated possible mechanisms leading to this outcome using an intratibial LNCaP model mimicking prostate cancer bone metastases. Male NOD.scid mice were inoculated intratibially with LNCaP prostate cancer cells and treated with vehicle, radium-223, abiraterone, prednisone, zoledronic acid, or their combinations for 28 days. Serum TRACP 5b and PSA levels were measured. Bone structure, quality, and formation rate of non-tumor-bearing and tumor-bearing tibiae were analyzed by microCT, 3-point bending assay, and dynamic histomorphometry, respectively. Radium-223 incorporation into bone was also measured. Radium-223/abiraterone/prednisone combination treatment induced a transient increase in bone resorption indicated by elevated TRACP 5b levels, which was inhibited by concurrent treatment with zoledronic acid. Furthermore, radium-223/abiraterone/prednisone combination reduced periosteal and trabecular new bone formation and the number of osteoblasts, but bone structure or biomechanical quality were not affected. The abiraterone/prednisone treatment decreased radium-223 incorporation into tumor-bearing bone, possibly explaining the lack of additional antitumor efficacy. In conclusion, radium-223/abiraterone/prednisone combination increased bone resorption, which may have been one of the mechanisms leading to an increased fracture risk in patients with mCRPC.

Characterization of Lymph Node Tumor Burden in Node-Positive Prostate Cancer Patients after Robotic-Assisted Radical Prostatectomy with Extended Pelvic Lymph Node Dissection.

BACKGROUND: Prostate cancer (PCa) nodal staging does not account for lymph node (LN) tumor burden. The LN anatomical compartment involved with the tumor or the quantified extent of extranodal extension (ENE) have not yet been studied in relation to biochemical recurrence-free survival (BRFS). METHODS: Histopathological slides of 66 pN1 PCa patients who underwent extended pelvic lymph node dissection were reviewed. We recorded metrics to quantify LN tumor burden. We also characterized the LN anatomical compartments involved and quantified the extent of ENE. RESULTS: The median follow-up time was 38 months. The median number of total LNs obtained per patient was 30 (IQR 23-37). In the risk-adjusted cox regression model, the following variables were associated with BRFS: mean size of the largest LN deposit per patient (log2: adjusted hazard ratio (aHR) = 1.91, p < 0.001), the mean total span of all LN deposits per patient (2.07, p < 0.001), and the mean percent surface area of the LN involved with the tumor (1.58, p < 0.001). There was no significant BRFS association for the LN anatomical compartment or the quantified extent of ENE. CONCLUSION: LN tumor burden is associated with BRFS. The LN anatomical compartments and the quantified extent of ENE did not show significant association with BRFS.

Linking folding dynamics and function of SAM/SAH riboswitches at the single molecule level.

Riboswitches are regulatory elements found in bacterial mRNAs that control downstream gene expression through ligand-induced conformational changes. Here, we used single-molecule FRET to map the conformational landscape of the translational SAM/SAH riboswitch and probe how co-transcriptional ligand-induced conformational changes affect its translation regulation function. Riboswitch folding is highly heterogeneous, suggesting a rugged conformational landscape that allows for sampling of the ligand-bound conformation even in the absence of ligand. The addition of ligand shifts the landscape, favoring the ligand-bound conformation. Mutation studies identified a key structural element, the pseudoknot helix, that is crucial for determining ligand-free conformations and their ligand responsiveness. We also investigated ribosomal binding site accessibility under two scenarios: pre-folding and co-transcriptional folding. The regulatory function of the SAM/SAH riboswitch involves kinetically favoring ligand binding, but co-transcriptional folding reduces this preference with a less compact initial conformation that exposes the Shine-Dalgarno sequence and takes min to redistribute to more compact conformations of the pre-folded riboswitch. Such slow equilibration decreases the effective ligand affinity. Overall, our study provides a deeper understanding of the complex folding process and how the riboswitch adapts its folding pattern in response to ligand, modulates ribosome accessibility and the role of co-transcriptional folding in these processes.

Structure and ion-dependent folding of k-junctions.

k-Junctions are elaborated forms of kink turns with an additional helix on the nonbulged strand, thus forming a three-way helical junction. Two were originally identified in the structures of Arabidopsis and Escherichia coli thiamine pyrophosphate (TPP) riboswitches, and another called DUF-3268 was tentatively identified from sequence information. In this work we show that the Arabidopsis and E. coli riboswitch k-junctions fold in response to the addition of magnesium or sodium ions, and that atomic mutations that should disrupt key hydrogen bonding interactions greatly impair folding. Using X-ray crystallography, we have determined the structure of the DUF-3268 RNA and thus confirmed that it is a k-junction. It also folds upon the addition of metal ions, though requiring a 40-fold lower concentration of either divalent or monovalent ions. The key difference between the DUF-3268 and riboswitch k-junctions is the lack of nucleotides inserted between G1b and A2b in the former. We show that this insertion is primarily responsible for the difference in folding properties. Finally, we show that the DUF-3268 can functionally substitute for the k-junction in the E. coli TPP riboswitch such that the chimera can bind the TPP ligand, although less avidly.

Biochemical and mechanistic analysis of the cleavage of branched DNA by human ANKLE1.

ANKLE1 is a nuclease that provides a final opportunity to process unresolved junctions in DNA that would otherwise create chromosomal linkages blocking cell division. It is a GIY-YIG nuclease. We have expressed an active domain of human ANKLE1 containing the GIY-YIG nuclease domain in bacteria, that is monomeric in solution and when bound to a DNA Y-junction, and unilaterally cleaves a cruciform junction. Using an AlphaFold model of the enzyme we identify the key active residues, and show that mutation of each leads to impairment of activity. There are two components in the catalytic mechanism. Cleavage rate is pH dependent, corresponding to a pKa of 6.9, suggesting an involvement of the conserved histidine in proton transfer. The reaction rate depends on the nature of the divalent cation, likely bound by glutamate and asparagine side chains, and is log-linear with the metal ion pKa. We propose that the reaction is subject to general acid-base catalysis, using a combination of tyrosine and histidine acting as general base and water directly coordinated to the metal ion as general acid. The reaction is temperature dependent; activation energy Ea = 37 kcal mol-1, suggesting that cleavage is coupled to opening of DNA in the transition state.

Multiple PIK3CA mutation clonality correlates with outcomes in taselisib + fulvestrant-treated ER+/HER2-, PIK3CA-mutated breast cancers.

BACKGROUND: Mutations in the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), encoded by the PIK3CA gene, cause dysregulation of the PI3K pathway in 35-40% of patients with HR+/HER2- breast cancer. Preclinically, cancer cells harboring double or multiple PIK3CA mutations (mut) elicit hyperactivation of the PI3K pathway leading to enhanced sensitivity to p110α inhibitors. METHODS: To understand the role of multiple PIK3CAmut in predicting response to p110α inhibition, we estimated the clonality of multiple PIK3CAmut in circulating tumor DNA (ctDNA) from patients with HR+/HER2- metastatic breast cancer enrolled to a prospectively registered clinical trial of fulvestrant ± taselisib, and analyzed the subgroups against co-altered genes, pathways, and outcomes. RESULTS: ctDNA samples with clonal multiple PIK3CAmut had fewer co-alterations in receptor tyrosine kinase (RTK) or non-PIK3CA PI3K pathway genes compared to samples with subclonal multiple PIK3CAmut indicating a strong reliance on the PI3K pathway. This was validated in an independent cohort of breast cancer tumor specimens that underwent comprehensive genomic profiling. Furthermore, patients whose ctDNA harbored clonal multiple PIK3CAmut exhibited a significantly higher response rate and longer progression-free survival vs subclonal multiple PIK3CAmut. CONCLUSIONS: Our study establishes clonal multiple PIK3CAmut as an important molecular determinant of response to p110α inhibition and provides rationale for further clinical investigation of p110α inhibitors alone or with rationally-selected therapies in breast cancer and potentially other solid tumor types.

Catalytic mechanism and pH dependence of a methyltransferase ribozyme (MTR1) from computational enzymology.

A methyltransferase ribozyme (MTR1) was selected in vitro to catalyze alkyl transfer from exogenous O6-methylguanine (O6mG) to a target adenine N1, and recently, high-resolution crystal structures have become available. We use a combination of classical molecular dynamics, ab initio quantum mechanical/molecular mechanical (QM/MM) and alchemical free energy (AFE) simulations to elucidate the atomic-level solution mechanism of MTR1. Simulations identify an active reactant state involving protonation of C10 that hydrogen bonds with O6mG:N1. The deduced mechanism involves a stepwise mechanism with two transition states corresponding to proton transfer from C10:N3 to O6mG:N1 and rate-controlling methyl transfer (19.4 kcal·mol-1 barrier). AFE simulations predict the pKa for C10 to be 6.3, close to the experimental apparent pKa of 6.2, further implicating it as a critical general acid. The intrinsic rate derived from QM/MM simulations, together with pKa calculations, enables us to predict an activity-pH profile that agrees well with experiment. The insights gained provide further support for a putative RNA world and establish new design principles for RNA-based biochemical tools.

Natural Antibodies Mediate Protection Against Acinetobacter baumannii Respiratory Infections.

BACKGROUND: Acinetobacter baumannii causes a wide range of dangerous infections due to the emergence of pandrug-resistant strains. Therefore, there is a need for alternative therapeutics to treat these infections, including those targeting the host immune responses. However, immune responses, especially the humoral response against this pathogen, are poorly understood. METHODS: This study investigated the lymphocyte-mediated innate immune resistance to A. baumannii AB5075 pulmonary infection using B- and T-cell-deficient (Rag2-/-) mice, the protective effect of natural antibodies (NAbs), and the expression of complement-mediated responses using a mouse pneumonia model. RESULTS: Our results showed that intranasally infected Rag2-/- mice are impaired in clearing bacteria from lung, liver, and spleen at 24 hours postinfection compared to wildtype mice. Animal pretreatment with normal mouse serum or purified antibodies from naive mice rescued Rag2-/- mice from infection. Analysis of C3 complement protein binding demonstrated that NAbs increased C3 protein deposition on A. baumannii cells, indicating the activation of the classical complement pathway by NAbs. CONCLUSIONS: Overall, our study shows that NAbs mediate innate immune resistance against A. baumannii, a finding that may lead to the development of effective therapies against human infections caused by this antibiotic-resistant A. baumannii.

Serum antibodies to peptidylarginine deiminase-4 in rheumatoid arthritis associated-interstitial lung disease are associated with decreased lung fibrosis and improved survival.

OBJECTIVE: Interstitial lung disease (ILD) is a leading cause of mortality in rheumatoid arthritis (RA), particularly in those with the usual interstitial pneumonia subtype (RA-UIP). Serum antibodies to peptidylarginine deiminase type 4 (anti-PAD4), particularly a subset that cross-react with PAD3 (PAD3/4XR), have been associated with imaging evidence of ILD. We aimed to determine the specificity of anti-PAD4 antibodies in RA-ILD and to examine associations with markers of ILD severity. METHODS: 48 RA-ILD and 31 idiopathic pulmonary fibrosis (IPF) patients were identified from the National Jewish Health Biobank. RA-ILD subtype was defined by imaging pattern on high-resolution chest computed tomography (CT), and serum was tested for anti-PAD4 and anti-PAD3/4XR antibodies. Antibody prevalence, measures of ILD severity (% predicted forced vital capacity, FVC; % predicted diffusion capacity carbon monoxide, DLCO; quantitative CT fibrosis) and mortality were compared between groups. RESULTS: Anti-PAD4 antibodies were present in 9/48 (19%) subjects with RA-ILD and no subjects with IPF. Within RA-ILD, anti-PAD4 antibodies were found almost exclusively in RA-UIP (89%). Within RA-UIP subjects, % predicted FVC was higher in anti-PAD4+ subjects, and this finding was most strongly associated with anti-PAD3/4XR antibodies. In addition, quantitative CT fibrosis score was lower in anti-PAD4+ RA-UIP subjects, including those with mono-reactive anti-PAD4 antibodies and anti-PAD3/4XR antibodies. Anti-PAD4+ RA-UIP subjects also exhibited decreased mortality. CONCLUSIONS: We demonstrate the presence of serum anti-PAD4 antibodies in a subset of patients with RA-UIP that were notably associated with better lung function, less fibrosis and decreased mortality.

ER+, HER2- advanced breast cancer treated with taselisib and fulvestrant: genomic landscape and associated clinical outcomes.

Taselisib is a potent ß-sparing phosphatidylinositol 3-kinase (PI3K) inhibitor that, with endocrine therapy, improves outcomes in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-mutated (PIK3CAmut) advanced breast cancer. To understand alterations associated with response to PI3K inhibition, we analysed circulating tumour DNA (ctDNA) from participants enrolled in the SANDPIPER trial. Participants were designated as either PIK3CAmut or PIK3CA no mutation was detected (NMD) per baseline ctDNA. The top mutated genes and tumour fraction estimates identified were analysed for their association with outcomes. In participants with PIK3CAmut ctDNA treated with taselisib + fulvestrant, tumour protein p53 (TP53; encoding p53) and fibroblast growth factor receptor 1 (FGFR1) alterations were associated with shorter progression-free survival (PFS) compared to participants with NMD in these genes. Conversely, participants with PIK3CAmut ctDNA harbouring a neurofibromin 1 (NF1) alteration or high baseline tumour fraction estimate experienced improved PFS upon treatment with taselisib + fulvestrant compared to placebo + fulvestrant. Broadly, alterations in oestrogen receptor (ER), PI3K and p53 pathway genes were associated with resistance to taselisib + fulvestrant in participants with PIK3CAmut ctDNA. Altogether, we demonstrated the impact of genomic (co-)alterations on outcomes with one of the largest clinico-genomic datasets of ER+, HER2-, PIK3CAmut breast cancer patients treated with a PI3K inhibitor.

Unicorns, Rhinoceroses and Chemical Bonds.

The nascent field of computationally aided molecular design will be built around the ability to make computation useful to synthetic chemists who draw on their empirically based chemical intuition to synthesize new and useful molecules. This fact poses a dilemma, as much of existing chemical intuition is framed in the language of chemical bonds, which are pictured as possessing physical properties. Unfortunately, it has been posited that calculating these bond properties is impossible because chemical bonds do not exist. For much of the computationalchemistry community, bonds are seen as mythical-the unicorns of the chemical world. Here, we show that this is not the case. Using the same formalism and concepts that illuminated the atoms in molecules, we shine light on the bonds that connect them. The real space analogue of the chemical bond becomes the bond bundle in an extended quantum theory of atoms in molecules (QTAIM). We show that bond bundles possess all the properties typically associated with chemical bonds, including an energy and electron count. In addition, bond bundles are characterized by a number of nontraditional attributes, including, significantly, a boundary. We show, with examples drawn from solid state and molecular chemistry, that the calculated properties of bond bundles are consistent with those that nourish chemical intuition. We go further, however, and show that bond bundles provide new and quantifiable insights into the structure and properties of molecules and materials.

Absolute Configuration Determination of Chiral API Molecules by MicroED Analysis of Cocrystal Powders Formed Based on Cocrystal Propensity Prediction Calculations.

Establishing the absolute configuration of chiral active pharmaceutical ingredients (APIs) is of great importance. Single crystal X-ray diffraction (scXRD) has traditionally been the method of choice for such analysis, but scXRD requires the growth of large crystals, which can be challenging. Here, we present a method for determining absolute configuration that does not rely on the growth of large crystals. By examining microcrystals formed with chiral probes (small chiral compounds such as amino acids), absolute configuration can be unambiguously determined by microcrystal electron diffraction (MicroED). Our streamlined method employs three steps: (1) virtual screening to identify promising chiral probes, (2) experimental cocrystal screening and (3) structure determination by MicroED and absolute configuration assignment. We successfully applied this method to analyze two chiral API molecules currently on the market for which scXRD was not used to determine absolute configuration.