MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

Feasibility of Tomotherapy-Based Image-Guided Radiotherapy to Reduce Aspiration Risk in Patients with Non-Laryngeal and Non-Pharyngeal Head and Neck Cancer

Purpose

The study aims to assess the feasibility of Tomotherapy-based image-guided radiotherapy (IGRT) to reduce the aspiration risk in patients with non-laryngeal and non-hypopharyngeal cancer. A retrospective review of 48 patients undergoing radiation for non-laryngeal and non-hypopharyngeal head and neck cancers was conducted. All patients had a modified barium swallow (MBS) prior to treatment, which was repeated one month following radiotherapy. Mean middle and inferior pharyngeal dose was recorded and correlated with the MBS results to determine aspiration risk.

Results

Mean pharyngeal dose was 23.2 Gy for the whole group. Two patients (4.2%) developed trace aspiration following radiotherapy which resolved with swallowing therapy. At a median follow-up of 19 months (1–48 months), all patients were able to resume normal oral feeding without aspiration.

Conclusion and Clinical Relevance

IGRT may reduce the aspiration risk by decreasing the mean pharyngeal dose in the presence of large cervical lymph nodes. Further prospective studies with IGRT should be performed in patients with non-laryngeal and non-hypopharyngeal head and neck cancers to verify this hypothesis.     

Feasibility of intensity-modulated and image-guided radiotherapy for functional organ preservation in locally advanced laryngeal cancer

Purpose

The study aims to assess the feasibility of intensity-modulated and image-guided radiotherapy (IMRT, and IGRT, respectively) for functional preservation in locally advanced laryngeal cancer. A retrospective review of 27 patients undergoing concurrent chemoradiation for locally advanced laryngeal cancers (8 IMRT, 19 IGRT) was undertaken. In addition to regular clinical examinations, all patients had PET imaging at 4 months and 10 months after radiotherapy, then yearly. Loco-regional control, speech quality and feeding-tube dependency were assessed during follow-up visits.

Results

At a median follow-up of 20 months (range 6–57 months), four out of 27 patients (14.8%) developed local recurrence and underwent salvage total laryngectomy. One patient developed distant metastases following salvage surgery. Among the 23 patients who conserved their larynx with no sign of recurrence at last follow-up, 22 (95%) reported normal or near normal voice quality, allowing them to communicate adequately. Four patients (14.8%) had long-term tube feeding-dependency because of severe dysphagia (2 patients) and chronic aspiration (2 patients, with ensuing death from aspiration pneumonia in one patient).

Conclusions and Clinical Relevance

Functional laryngeal preservation is feasible with IMRT and IGRT for locally advanced laryngeal cancer. However, dysphagia and aspiration remain serious complications, due most likely to high radiation dose delivery to the pharyngeal musculatures.     

Differential Subcellular Distributions and Trafficking Functions of hnRNP A2/B1 Spliceoforms

Trafficking of mRNA molecules from the nucleus to distal processes in neural cells is mediated by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 trans‐acting factors. Although hnRNP A2/B1 is alternatively spliced to generate four isoforms, most functional studies have not distinguished between these isoforms. Here, we show, using isoform‐specific antibodies and isoform‐specific green fluorescent protein (GFP)‐fusion expression constructs, that A2b is the predominant cytoplasmic isoform in neural cells, suggesting that it may play a key role in mRNA trafficking. The differential subcellular distribution patterns of the individual isoforms are determined by the presence or absence of alternative exons that also affect their dynamic behavior in different cellular compartments, as measured by fluorescence correlation spectroscopy. Expression of A2b is also differentially regulated with age, species and cellular development. Furthermore, coinjection of isoform‐specific antibodies and labeled RNA into live oligodendrocytes shows that the assembly of RNA granules is impaired by blockade of A2b function. These findings suggest that neural cells modulate mRNA trafficking by regulating alternative splicing of hnRNP A2/B1 and controlling expression levels of A2b, which may be the predominant mediator of cytoplasmic‐trafficking functions. These findings highlight the importance of considering isoform‐specific functions for alternatively spliced proteins.     

Feasibility of Image-Guided Radiotherapy for Elderly Patients with Locally Advanced Rectal Cancer

Purpose

The study aims to assess the tolerance of elderly patients (70 years or older) with locally advanced rectal cancers to image-guided radiotherapy (IGRT). A retrospective review of 13 elderly patients with locally advanced rectal cancer who underwent preoperative chemoradiation using IGRT was performed. Grade 3–4 acute toxicities, survival, and long-term complications were compared to 17 younger patients (<70 years) with the same disease stage.

Results

Grade 3–4 hematologic toxicities occurred in 7.6% and 0% (p = 0.4) and gastrointestinal toxicities, and, in 15.2% and 5% (p = 0.5), of elderly and younger patients, respectively. Surgery was aborted in three patients, two in the elderly group and one in the younger group. One patient in the elderly group died after surgery from cardiac arrhythmia. After a median follow-up of 34 months, five patients had died, two in the elderly and three in the younger group. The 3-year survival was 90.9% and 87.5% (p = 0.7) for the elderly and younger group respectively. Two patients in the younger group developed ischemic colitis and fecal incontinence. There was no statistically significant difference in acute and late toxicities as well as survival between the two groups.

Conclusions and Clinical Relevance

Elderly patients with locally advanced rectal cancers may tolerate preoperative chemoradiation with IGRT as well as younger patients. Further prospective studies should be performed to investigate the potential of IGRT for possible cure in elderly patients with locally advanced rectal cancer.     

The effects of a transcontinental flight on markers of coagulation and fibrinolysis in healthy men after vigorous physical activity

Purpose: Athletes and military service members are known to undergo strenuous exercise and sometimes have to take long haul flights soon afterwards; however, its combined effect on many physiological functions is relatively unknown. Therefore, we examined the combined effects of a full-body muscle-damaging workout and transcontinental flight on coagulation and fibrinolysis in healthy, resistance trained men. We also determined the efficacy of a full-body compression garment in limiting their coagulation responses. Materials and Methods: Nineteen healthy, resistance trained men flew from Connecticut (CT) to California (CA), performed a full-body muscle-damaging workout and then flew back to CT. Ten participants wore full-body compression garments (FCG) for the duration of both flights and during all other portions of the study except during workouts and blood draws, when they wore loose clothing. Nine controls wore loose clothing (CON) throughout the study. Blood samples were collected at 16 h and 3 h before the initial flight from CT, immediately after landing in CA, immediately before and immediately after the full-body workout in CA, immediately after landing in CT, and at 29 h after landing in CT. Plasma markers of coagulation included activated partial thromboplastin time (aPTT), prothrombin fragment 1+2 (PTF 1+2) and thrombin ant-thrombin (TAT). Markers of the fibrinolytic system included the tissue plasmigen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and D-Dimer. Results: Both FCG and CON groups exhibited a faster aPTT after the full-body workout compared to all other time points. Thrombin generation markers, TAT and PTF 1+2, increased significantly after the full-body workout and immediately after landing in CT. Additionally, tPA increased after the full-body workout, while PAI-1 increased before the flight to CA, after the full-body workout, and just after landing in CT. The D-Dimer significantly increased after the full-body workout and at 29 h post-flight in both groups. Between groups, aPTT was significantly faster and TAT elevated with the CON group at 29 h post-flight. Also, PAI-1 demonstrated higher concentrations immediately after landing in CT for the CON group. Conclusion: A full-body muscle-damaging workout in conjunction with a trans-continental flight activated the coagulation and fibrinolytic systems. Additionally, wearing a full-body compression garment may limit coagulation following a workout through the recovery period.     

Feasibility of Tomotherapy-Based Image-Guided Radiotherapy for Locally Advanced Oropharyngeal Cancer

Purpose

The study aims to assess the feasibility of tomotherapy-based image-guided (IGRT) radiotherapy for locally advanced oropharyngeal cancer. A retrospective review of 33 patients undergoing concurrent chemoradiation for locally advanced oropharyngeal cancers was conducted. Radiotherapy planning, treatment toxicity and loco-regional control were assessed.

Results

At a median follow-up of 32 months (6–47 months), no patient developed loco-regional recurrence. Two patients (6%) developed distant metastases. Grade 3–4 acute toxicity was respectively 72% and 25% for mucositis and gastrointestinal toxicity. Two patients (6%) had long-term dependence on tube feedings. Dose-volume histogram demonstrated excellent target volume coverage and low radiation dose to the organs at risk for complications.

Conclusions and clinical relevance

IGRT provides excellent loco-regional control but acute toxicity remains significant and needs to be addressed in future prospective trials. The feasibility of Tomotherapy to decrease radiation dose to the normal tissues merits further investigations.