Comparison of early surgical outcomes of robotic and laparoscopic colorectal cancer resection reported by a busy district general hospital in England.

Robotic platforms provide a stable tool with high-definition views and improved ergonomics compared to laparoscopic approaches. The aim of this retrospective study was to compare the intra- and short-term postoperative results of oncological resections performed robotically (RCR) and laparoscopically (LCR) at a single centre. Between February 2020 and October 2022, retrospective data on RCR were compared to LCR undertaken during the same period. Parameters compared include total operative time, length of stay (LOS), re-admission rates, 30-day morbidity. 100 RCR and 112 LCR satisfied inclusion criteria. There was no difference between the two group's demographic and tumour characteristics. Overall, median operative time was shorter in LCR group [200 vs. 247.5 min, p < 0.005], but this advantage was not observed with pelvic and muti-quadrant resections. There was no difference in the rate of conversion [5(5%) vs. 5(4.5%), p > 0.95]. With respect to perioperative outcomes, there was no difference in the overall morbidity, or mortality between RCR and LCR, in particular requirement for blood transfusion [3(3%) vs. 5(4.5%), p 0.72], prolonged ileus [9(9%) vs. 15(13.2%), p 0.38], surgical site infections [5(4%) vs. 5(4.4%), p > 0.95], anastomotic leak [7(7%) vs. 5(4.4%), p 0.55], and re-operation rate [9(9%) vs. 7(6.3%), p 0.6]. RCR had shorter LOS by one night, but this did not reach statistical significance. No difference was observed in completeness of resection but there was a statically significant increase in lymph node harvest in the robotic series. Robotic approach to oncological colorectal resections is safe, with comparable intra- and peri-operative morbidity and mortality to laparoscopic surgery.

Laparoscopic right hemicolectomy with complete mesocolic excision and D3 lymphadenectomy using the open book approach: a video vignette.

According to the concept of total mesorectal excision for rectal cancer, Hohenberger translated this concept to colonic cancer by introducing complete mesocolic excision (CME). The concept of this surgical technique was further elucidated by Benz et al. in the form of an open book approach. This article presents and demonstrates in a video a case of laparoscopic right hemicolectomy with CME and D3 lymphadenectomy using open book approach in the treatment of a T3N1M0 distal ascending colonic adenocarcinoma. The final pathology report confirmed moderately differentiated adenocarcinoma with a maximum tumor size of 55 mm and 0/60 lymph nodes. The mesocolic fascia was intact and R0 was achieved. The final staging was pT3pN0pM0. However, D3 lymphadenectomy is not universally adopted due to concerns of higher morbidity we believe that with adequate training and supervision CME with D3 LDN is feasible and safe to be offered to all right-sided colorectal cancers with curative intent treatment.

Degradation of neurodegenerative disease-associated TDP-43 aggregates and oligomers via a proteolysis-targeting chimera.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) associated with TAR DNA-binding protein 43 (TDP-43) aggregation has been considered as a lethal and progressive motor neuron disease. Recent studies have shown that both C-terminal TDP-43 (C-TDP-43) aggregates and oligomers were neurotoxic and pathologic agents in ALS and frontotemporal lobar degeneration (FTLD). However, misfolding protein has long been considered as an undruggable target by applying conventional inhibitors, agonists, or antagonists. To provide this unmet medical need, we aim to degrade these misfolding proteins by designing a series of proteolysis targeting chimeras (PROTACs) against C-TDP-43. METHODS: By applying filter trap assay, western blotting, and microscopy imaging, the degradation efficiency of C-TDP-43 aggregates was studied in Neuro-2a cells overexpressing eGFP-C-TDP-43 or mCherry-C-TDP-43. The cell viability was characterized by alarmarBlue assay. The beneficial and disaggregating effects of TDP-43 PROTAC were examined with the YFP-C-TDP-43 transgenic C. elegans by motility assay and confocal microscopy. The impact of TDP-43 PROTAC on C-TDP-43 oligomeric intermediates was monitored by fluorescence lifetime imaging microscopy and size exclusion chromatography in the Neuro-2a cells co-expressing eGFP-C-TDP-43 and mCherry-C-TDP-43. RESULTS: Four PROTACs with different linker lengths were synthesized and characterized. Among these chimeras, PROTAC 2 decreased C-TDP-43 aggregates and relieved C-TDP-43-induced cytotoxicity in Neuro-2a cells without affecting endogenous TDP-43. We showed that PROTAC 2 bound to C-TDP-43 aggregates and E3 ligase to initiate ubiquitination and proteolytic degradation. By applying advanced microscopy, it was further shown that PROTAC 2 decreased the compactness and population of C-TDP-43 oligomers. In addition to cellular model, PROTAC 2 also improved the motility of transgenic C. elegans by reducing the C-TDP-43 aggregates in the nervous system. CONCLUSIONS: Our study demonstrated the dual-targeting capacity of the newly-designed PROTAC 2 against both C-TDP-43 aggregates and oligomers to reduce their neurotoxicity, which shed light on the potential drug development for ALS as well as other neurodegenerative diseases.

Polyglutamine-Specific Gold Nanoparticle Complex Alleviates Mutant Huntingtin-Induced Toxicity.

Huntington's disease (HD) belongs to protein misfolding disorders associated with polyglutamine (polyQ)-rich mutant huntingtin (mHtt) protein inclusions. Currently, it is indicated that the aggregation of polyQ-rich mHtt participates in neuronal toxicity and dysfunction. Here, we designed and synthesized a polyglutamine-specific gold nanoparticle (AuNP) complex, which specifically targeted mHtt and alleviated its toxicity. The polyglutamine-specific AuNPs were prepared by decorating the surface of AuNPs with an amphiphilic peptide (JLD1) consisting of both polyglutamine-binding sequences and negatively charged sequences. By applying the polyQ aggregation model system, we demonstrated that AuNPs-JLD1 dissociated the fibrillary aggregates from the polyQ peptide and reduced its ß-sheet content in a concentration-dependent manner. By further integrating polyethyleneimine (PEI) onto AuNPs-JLD1, we generated a complex (AuNPs-JLD1-PEI). We showed that this complex could penetrate cells, bind to cytosolic mHtt proteins, dissociate mHtt inclusions, reduce mHtt oligomers, and ameliorate mHtt-induced toxicity. AuNPs-JLD1-PEI was also able to be transported to the brain and improved the functional deterioration in the HD Drosophila larva model. Our results revealed the feasibility of combining AuNPs, JLD1s, and cell-penetrating polymers against mHtt protein aggregation and oligomerization, which hinted on the early therapeutic strategies against HD.

The Different Faces of the TDP-43 Low-Complexity Domain: The Formation of Liquid Droplets and Amyloid Fibrils.

Transactive response DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein that is involved in transcription and translation regulation, non-coding RNA processing, and stress granule assembly. Aside from its multiple functions, it is also known as the signature protein in the hallmark inclusions of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) patients. TDP-43 is built of four domains, but its low-complexity domain (LCD) has become an intense research focus that brings to light its possible role in TDP-43 functions and involvement in the pathogenesis of these neurodegenerative diseases. Recent endeavors have further uncovered the distinct biophysical properties of TDP-43 under various circumstances. In this review, we summarize the multiple structural and biochemical properties of LCD in either promoting the liquid droplets or inducing fibrillar aggregates. We also revisit the roles of the LCD in paraspeckles, stress granules, and cytoplasmic inclusions to date.

FDG-PET/CT in colorectal cancer: potential for vascular-metabolic imaging to provide markers of prognosis.

PURPOSE: This study assesses the potential for vascular-metabolic imaging with FluoroDeoxyGlucose (FDG)-Positron Emission Tomography/Computed Tomography (PET/CT) perfusion to provide markers of prognosis specific to the site and stage of colorectal cancer. METHODS: This prospective observational study comprised of participants with suspected colorectal cancer categorized as either (a) non-metastatic colon cancer (M0colon), (b) non-metastatic rectal cancer (M0rectum), or (c) metastatic colorectal cancer (M+). Combined FDG-PET/CT perfusion imaging was successfully performed in 286 participants (184 males, 102 females, age: 69.60 ± 10 years) deriving vascular and metabolic imaging parameters. Vascular and metabolic imaging parameters alone and in combination were investigated with respect to overall survival. RESULTS: A vascular-metabolic signature that was significantly associated with poorer survival was identified for each patient group: M0colon - high Total Lesion Glycolysis (TLG) with increased Permeability Surface Area Product/Blood Flow (PS/BF), Hazard Ratio (HR) 3.472 (95% CI: 1.441-8.333), p = 0.006; M0rectum - high Metabolic Tumour Volume (MTV) with increased PS/BF, HR 4.567 (95% CI: 1.901-10.970), p = 0.001; M+ participants, high MTV with longer Time To Peak (TTP) enhancement, HR 2.421 (95% CI: 1.162-5.045), p = 0.018. In participants with stage 2 colon cancer as well as those with stage 3 rectal cancer, the vascular-metabolic signature could stratify the prognosis of these participants. CONCLUSION: Vascular and metabolic imaging using FDG-PET/CT can be used to synergise prognostic markers. The hazard ratios suggest that the technique may have clinical utility.

Nanoscopic investigation of C9orf72 poly-GA oligomers on nuclear membrane disruption by a photoinducible platform.

Glycine-alanine dipeptide repeats (GA DPRs) translated from the mutated C9orf72 gene have recently been correlated with amyotrophic lateral sclerosis (ALS). While GA DPRs aggregates have been suggested as amyloid, the biophysical features and cytotoxicity of GA DPRs oligomers has not been explored due to its unstable nature. In this study, we develop a photoinducible platform based on methoxynitrobenzene chemistry to enrich GA DPRs that allows monitoring the oligomerization process of GA DPRs in cells. By applying advanced microscopies, we examined the GA DPRs oligomerization process nanoscopically in a time-dependent manner. We provided direct evidences to demonstrate GA DPRs oligomers rather than nanofibrils disrupt nuclear membrane. Moreover, we found GA DPRs hamper nucleocytoplasmic transport in cells and cause cytosolic retention of TAR DNA-binding protein 43 in cortical neurons. Our results highlight the toxicity of GA DPRs oligomers, which is a key step toward elucidating the pathological roles of C9orf72 DPRs.

Nanoscopic Insights of Amphiphilic Peptide against the Oligomer Assembly Process to Treat Huntington's Disease.

Finding an effective therapeutic regimen is an urgent demand for various neurodegenerative disorders including Huntington's disease (HD). For the difficulties in observing the dynamic aggregation and oligomerization process of mutant Huntingtin (mHtt) in vivo, the evaluation of potential drugs at the molecular protein level is usually restricted. By combing lifetime-based fluorescence microscopies and biophysical tools, it is showcased that a designed amphiphilic peptide, which targets the mHtt at an early stage, can perturb the oligomer assembly process nanoscopically, suppress the amyloid property of mHtt, conformationally transform the oligomers and/or aggregates of mHtt, and ameliorate mHtt-induced neurological damage and aggregation in cell and HD mouse models. It is also found that this amphiphilic peptide is able to transport to the brain and rescue the memory deficit through intranasal administration, indicating its targeting specificity in vivo. In summary, a biophotonic platform is provided to investigate the oligomerization/aggregation process in detail that offers insight into the design and effect of a targeted therapeutic agent for Huntington's disease.

Single Port Laparoscopic Total and Subtotal Colectomies for Inflammatory Bowel Disease in a District General Hospital.

Background: Single incision laparoscopic surgery (SILS) is expanding, enhancing the advantages of multi-port laparoscopic surgery (MLS). Limited literature exists regarding SILS total/subtotal colectomies for inflammatory bowel disease (IBD). Aim of the study was to present the initial experience with this type of approach in a district general hospital and extrapolate its feasibility and safety in this specific context based on gold standard outcomes reported in literature. Materials and Methods: Preoperative parameters, operative details and surgical outcomes of consecutive patients who underwent colonic SILS for IBD in a 5-year period were reviewed retrospectively. Median length of follow-up was 26 months. Results: Fourteen patients underwent SILS subtotal/total colectomy. Median body mass index was 25 (18.1-35). Two patients had previous abdominal surgeries. Median operating time was 202.5 minutes. Two cases were converted to open. Median length of stay was 5 days. Three patients presented complications. Three patients developed parastomal hernias (21.4%). Five out of 12 patients with ulcerative colitis declined further surgery, 3 are awaiting laparoscopic/SILS pouch formation, 1 underwent SILS pouch formation, 1 SILS ileo-rectal anastomosis and 1 patient had SILS completion proctectomy. One patient was not followed up. Conclusions: Despite literature data heterogeneity, these results provide support to the feasibility and applicability of SILS in the subgroup of patients who undergo subtotal/total colectomies for IBD, offering the option for subsequent SILS completion or restorative procedures. Further studies are required to explore the benefit of SILS over MLS (including cosmesis and quality of life) and non-inferiority of SILS regarding the parastomal hernia issue and the operative duration.

Biocompatible Inhibitor Based on Chitosan and Amphiphilic Peptide against Mutant Huntingtin Toxicity.

Huntington's disease (HD) is classified as a protein-misfolding disease correlated with the mutant Huntingtin (mHtt) protein with abnormally expanded polyglutamine (polyQ) domains. Because no effective drugs have yet been reported, attempts to develop better therapy to delay the age of onset are in urgent demand. In this study, an amphiphilic peptide consisting of negatively charged hexaglutamic acid and a stretch of decaglutamine (E6 Q10 ) was chemically synthesized as an inhibitor against polyQ and mHtt toxicity. It is found that E6 Q10 selfassembles into spherical vesicles, as shown by means of TEM, cryoelectron microscopy, and dynamic light scattering. Assembled E6 Q10 prevented the polyQ-rich peptide (KKWQ20 AKK) from forming amyloid fibrils. To enable the cell-penetration ability of E6 Q10 , the E6 Q10 ⋅chitosan complex was generated. It is demonstrated that the complex penetrates cells, interferes with the mHtt oligomerization and aggregation process, and prevents mHtt cytotoxicity. By combining positively charged chitosan and amphiphilic peptides with a negatively charge moiety, a new strategy is provided to develop biocompatible and biodegradable inhibitors against mHtt toxicity.

Viral Cre-LoxP tools aid genome engineering in mammalian cells.

BACKGROUND: Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. RESULTS: We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions. CONCLUSIONS: We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.

Photocontrollable Probe Spatiotemporally Induces Neurotoxic Fibrillar Aggregates and Impairs Nucleocytoplasmic Trafficking.

The abnormal assembly of misfolded proteins into neurotoxic aggregates is the hallmark associated with neurodegenerative diseases. Herein, we establish a photocontrollable platform to trigger amyloidogenesis to recapitulate the pathogenesis of amyotrophic lateral sclerosis (ALS) by applying a chemically engineered probe as a "switch" in live cells. This probe is composed of an amyloidogenic peptide from TDP-43, a photolabile linker, a polycationic sequence both to mask amyloidogenicity and for cell penetration, and a fluorophore for visualization. The photocontrollable probe can self-assemble into a spherical vesicle but rapidly develops massive nanofibrils with amyloid properties upon photoactivation. The photoinduced in vitro fibrillization process is characterized by biophysical techniques. In cellular experiments, this cell-penetrable vesicle was retained in the cytoplasm, seeded the mislocalized endogenous TDP-43 into aggregates upon irradiation, and consequently initiated apoptosis. In addition, this photocontrollable vesicle interfered with nucleocytoplasmic protein transport and triggered cortical neuron degeneration. Our developed strategy provides in vitro and in vivo spatiotemporal control of neurotoxic fibrillar aggregate formation, which can be readily applied in the studies of protein misfolding, aggregation-induced protein mislocalization, and amyloid-induced pathogenesis in different diseases.

Trigger Factor-Induced Nascent Chain Dynamics Changes Suggest Two Different Chaperone-Nascent Chain Interactions during Translation.

Protein biogenesis is poorly understood due to the ribosome that perturbs measurement attempted on the ribosome-bound nascent chain (RNC). Investigating nascent chain dynamics may provide invaluable insight into the co-translational processes such as structure formation or interaction with a chaperone [e.g., the bacterial trigger factor (TF)]. In this study, we aim to establish a platform for studying nascent chain dynamics by exploring the local environment near the fluorescent dye on site-specifically labeled RNCs with time-resolved fluorescence anisotropy. To prepare a quantitative model of fluorescence depolarization, we utilized intrinsically disordered protein bound to ribosome, which helped us couple the sub-nanosecond depolarization with the motion of the nascent chain backbone. This was consistent with zinc-finger-domain-containing RNCs, where the extent of sub-nanosecond motion decreased upon the addition of zinc when the fluorophore was in close proximity of the domain. After the characterization of disordered nascent chain dynamics, we investigated the synthesis of a model cytosolic protein, Entner-Doudoroff aldolase, labeled at different sites during various stages of translation. Depending on the stage of translation, the addition of the TF to the nascent chain led to two different responses in the nascent chain dynamics serendipitously, suggesting steric hindrance between the nascent chain and the chaperone as a mechanism for TF dissociation from the ribosome during translation. Overall, our study demonstrates the possible use of site-specific labeling and time-resolved anisotropy to gain insight on chaperone binding event at various stages of translation and hints on TF co-translational mechanism.

Study of Binding Interaction between Pif80 Protein Fragment and Aragonite.

Pif is a crucial protein for the formation of the nacreous layer in Pinctada fucata. Three non-acidic peptide fragments of the aragonite-binding domain (Pif80) are selected, which contain multiple copies of the repeat sequence DDRK, to study the interaction between non-acidic peptides and aragonite. The polypeptides DDRKDDRKGGK (Pif80-11) and DDRKDDRKGGKDDRKDDRKGGK (Pif80-22) have similar binding affinity to aragonite. Solid-state NMR data indicate that the backbones of Pif80-11 and Pif80-22 peptides bound on aragonite adopt a random-coil conformation. Pif80-11 is a lot more effective than Pif80-22 in promoting the nucleation of aragonite on the substrate of ß-chitin. Our results suggest that the structural arrangement at a protein-mineral interface depends on the surface structure of the mineral substrate and the protein sequence. The side chains of the basic residues, which function as anchors to the aragonite surface, have uniform structures. The role of basic residues as anchors in protein-mineral interaction may play an important role in biomineralization.

An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus.

BACKGROUND: A number of integrase defective lentiviral (IDLV) packaging systems have been developed to produce integration deficient lentiviruses for gene delivery and epichromosomal expression. However, despite their growing demand, a comparative study to systemically evaluate the performance efficiency of different mutants on virus packaging and gene expression has not been done. RESULTS: Site-directed mutagenesis was used to generate five integrasedeficient mutants for non-integrative lentiviral packaging (NILVP). The five mutants were then individually incorporated to make different integrase defective lentivirus plasmid packaging mix, keeping other packaging factors constant. CD511B-1, a lentivectorexpressing GFP from an EF1 promoter, was packaged with each of the five different lentivirus packaging mix to make pseudotypedviral particles. The performance and packaging efficiency of each of the integrase deficient mutants was evaluated based on GFP expression in HT1080 cells, while the wild type lentivirus packaging mix was used as a control. Of the five integrase mutant candidates, one with the highestGFP transgene expression level was chosen for further characterization. The non-integrative nature of this candidate was confirmed by quantitative polymerase chain reaction and characterized using both dividing and non-dividing cells. Finally, a detailed standard protocol for NILVP using this integrase defective mutant was developed. CONCLUSIONS: An efficient lentiviral packaging system for producing on-integrative lentivirus was established. This system is compatible with most existing lentivectors and can be used to transduce both dividing and non-dividing cells.

Painful, swollen, oozing right great toe.

We initially suspected septic arthritis or osteomyelitis in this patient. Further investigation proved it was neither.

Mechanisms of Membrane Pore Formation by Amyloidogenic Peptides in Amyotrophic Lateral Sclerosis.

Using unbiased atomic-detailed molecular dynamics simulations, the C-terminal fragments of TDP-43 are observed to aggregate and form disordered-toroidal pores in a lipid bilayer. Cytotoxicity of TDP-43 may be inferred from the observation that the membrane pores catalyze lipid flip-flop between bilayer leaflets and conduct water at high rates.

Conformational switch of polyglutamine-expanded huntingtin into benign aggregates leads to neuroprotective effect.

The abundant accumulation of inclusion bodies containing polyglutamine-expanded mutant huntingtin (mHTT) aggregates is considered as the key pathological event in Huntington's disease (HD). Here, we demonstrate that FKBP12, an isomerase that exhibits reduced expression in HD, decreases the amyloidogenicity of mHTT, interrupts its oligomerization process, and structurally promotes the formation of amorphous deposits. By combining fluorescence-activated cell sorting with multiple biophysical techniques, we confirm that FKBP12 reduces the amyloid property of these ultrastructural-distinct mHTT aggregates within cells. Moreover, the neuroprotective effect of FKBP12 is demonstrated in both cellular and nematode models. Finally, we show that FKBP12 also inhibit the fibrillization process of other disease-related and aggregation-prone peptides. Our results suggest a novel function of FKBP12 in ameliorating the proteotoxicity in mHTT, which may shed light on unraveling the roles of FKBP12 in different neurodegenerative diseases and developing possible therapeutic strategies.