High-Resolution Intact Protein Analysis via Phase-Modulated, Stepwise Frequency Scan Ion Trap Mass Spectrometry.

Mass spectrometry (MS) using an electron multiplier for intact protein analysis remains limited. Because of the massive size and complex structure of proteins, the slow flight speed of their ions results in few secondary electrons and thus low detection sensitivity and poor spectral resolution. Thus, we present a compact ion trap-mass spectrometry approach to directly detect ion packets and obtain the high-resolution molecular signature of proteins. The disturbances causing deviations of ion motion and mass conversion have been clarified in advance. The radio frequency waveform used to manipulate ions is proposed to be a sequence of constant-frequency steps, interconnected by short time-outs, resulting in least dispersive distortion. Furthermore, more such constant-phase conjunctions are arranged in each step to compensate for fluctuations resulting from defects in the system and operation. In addition, two auxiliary pulses are generated in the right phase of each step to select ions of a specific secular state to detect one clean and sharp spectral line.This study demonstrates a top-down approach for the MS measurement of cytochrome C molecules, resulting in a spectral profile of the protein in its natural state at a resolution of 20 Da. Additionally, quick MS scans of other proteins were performed.

AB092. Imaging the histopathology subtypes of the growth hormone-secreting pituitary neuroendocrine tumor.

BACKGROUND: Sparsely granulated (SG) growth hormone-secreting pituitary neuroendocrine tumors (GH-PitNETs) often present with a more aggressive clinical course compared to densely granulated (DG) tumors. These subtypes exhibit distinct biological and imaging characteristics. Thus, this study aims to differentiate between the histopathological subtypes of GH-PitNETs using pre-operative magnetic resonance imaging (MRI). METHODS: A retrospective analysis was conducted on 83 acromegalic patients treated at our institution between 2000 and 2010. Tumor volumes were segmented from preoperative MRIs, including T1-weighted, T2-weighted, T1 with contrast, and T2 fluid attenuated inversion recovery (FLAIR) sequences. Reference regions of interest (ROIs) were delineated using gray and white matter from the same sequences. Two pathologists reviewed pathology specimens for anti-cytokeratin (CAM 5.2) and Pit-1 expression. Clinical and radiological biomarkers were compared between SG and DG patients. RESULTS: A total of 83 patients with complete histopathology and 51 patients with complete MRIs were included in the analysis. SG PitNETs exhibited higher rates of supra-sellar invasion (61.5%, P<0.001), larger tumor sizes, lower pre-operative GH levels, and increased post-operative residual tumor (65.4%, P<0.001) compared to DG PitNETs. Additionally, SG PitNETs showed greater hyperintensity on T2-weighted images and enhanced contrast, whereas DG PitNETs exhibited less contrast enhancement. Utilization of these imaging biomarkers demonstrated an 94.1% accuracy in T2 FLAIR and overall of 78.7% predicting the histopathological subtypes of GH-PitNETs. CONCLUSIONS: Distinct histopathological subtypes of GH-PitNETs represent crucial prognostic factors. Utilizing multimodal pre-operative MRIs, clinicians can accurately identify sparsely granulated GH-PitNETs, facilitating improved treatment planning strategies.

Dual protection by Bcp1 and Rkm1 ensures incorporation of uL14 into pre-60S ribosomal subunits.

Eukaryotic ribosomal proteins contain extended regions essential for translation coordination. Dedicated chaperones stabilize the associated ribosomal proteins. We identified Bcp1 as the chaperone of uL14 in Saccharomyces cerevisiae. Rkm1, the lysine methyltransferase of uL14, forms a ternary complex with Bcp1 and uL14 to protect uL14. Rkm1 is transported with uL14 by importins to the nucleus, and Bcp1 disassembles Rkm1 and importin from uL14 simultaneously in a RanGTP-independent manner. Molecular docking, guided by crosslinking mass spectrometry and validated by a low-resolution cryo-EM map, reveals the correlation between Bcp1, Rkm1, and uL14, demonstrating the protection model. In addition, the ternary complex also serves as a surveillance point, whereas incorrect uL14 is retained on Rkm1 and prevented from loading to the pre-60S ribosomal subunits. This study reveals the molecular mechanism of how uL14 is protected and quality checked by serial steps to ensure its safe delivery from the cytoplasm until its incorporation into the 60S ribosomal subunit.

REDOMA: Bayesian random-effects dose-optimization meta-analysis using spike-and-slab priors.

The rise of cutting-edge precision cancer treatments has led to a growing significance of the optimal biological dose (OBD) in modern oncology trials. These trials now prioritize the consideration of both toxicity and efficacy simultaneously when determining the most desirable dosage for treatment. Traditional approaches in early-phase oncology trials have conventionally relied on the assumption of a monotone relationship between treatment efficacy and dosage. However, this assumption may not hold valid for novel oncology therapies. In reality, the dose-efficacy curve of such treatments may reach a plateau at a specific dose, posing challenges for conventional methods in accurately identifying the OBD. Furthermore, achieving reliable identification of the OBD is typically not possible based on a single small-sample trial. With data from multiple phase I and phase I/II trials, we propose a novel Bayesian random-effects dose-optimization meta-analysis (REDOMA) approach to identify the OBD by synthesizing toxicity and efficacy data from each trial. The REDOMA method can address trials with heterogeneous characteristics. We adopt a curve-free approach based on a Gamma process prior to model the average dose-toxicity relationship. In addition, we utilize a Bayesian model selection framework that uses the spike-and-slab prior as an automatic variable selection technique to eliminate monotonic constraints on the dose-efficacy curve. The good performance of the REDOMA method is confirmed by extensive simulation studies.

On the relative conservativeness of Bayesian logistic regression method in oncology dose-finding studies.

The Bayesian logistic regression method (BLRM) is a widely adopted and flexible design for finding the maximum tolerated dose in oncology phase I studies. However, the BLRM design has been criticized in the literature for being overly conservative due to the use of the overdose control rule. Recently, a discussion paper titled "Improving the performance of Bayesian logistic regression model with overall control in oncology dose-finding studies" in Statistics in Medicine has proposed an overall control rule to address the "excessive conservativeness" of the standard BLRM design. In this short communication, we discuss the relative conservativeness of the standard BLRM design and also suggest a dose-switching rule to further enhance its performance.

A unified view on enzyme catalysis by cryo-EM study of a DNA topoisomerase.

The theories for substrate recognition in enzyme catalysis have evolved from lock-key to induced fit, then conformational selection, and conformational selection followed by induced fit. However, the prevalence and consensus of these theories require further examination. Here we use cryogenic electron microscopy and African swine fever virus type 2 topoisomerase (AsfvTop2) to demonstrate substrate binding theories in a joint and ordered manner: catalytic selection by the enzyme, conformational selection by the substrates, then induced fit. The apo-AsfvTop2 pre-exists in six conformers that comply with the two-gate mechanism directing DNA passage and release in the Top2 catalytic cycle. The structures of AsfvTop2-DNA-inhibitor complexes show that substantial induced-fit changes occur locally from the closed apo-conformer that however is too far-fetched for the open apo-conformer. Furthermore, the ATPase domain of AsfvTop2 in the MgAMP-PNP-bound crystal structures coexist in reduced and oxidized forms involving a disulfide bond, which can regulate the AsfvTop2 function.

A structure of the relict phycobilisome from a thylakoid-free cyanobacterium.

Phycobilisomes (PBS) are antenna megacomplexes that transfer energy to photosystems II and I in thylakoids. PBS likely evolved from a basic, inefficient form into the predominant hemidiscoidal shape with radiating peripheral rods. However, it has been challenging to test this hypothesis because ancestral species are generally inaccessible. Here we use spectroscopy and cryo-electron microscopy to reveal a structure of a "paddle-shaped" PBS from a thylakoid-free cyanobacterium that likely retains ancestral traits. This PBS lacks rods and specialized ApcD and ApcF subunits, indicating relict characteristics. Other features include linkers connecting two chains of five phycocyanin hexamers (CpcN) and two core subdomains (ApcH), resulting in a paddle-shaped configuration. Energy transfer calculations demonstrate that chains are less efficient than rods. These features may nevertheless have increased light absorption by elongating PBS before multilayered thylakoids with hemidiscoidal PBS evolved. Our results provide insights into the evolution and diversification of light-harvesting strategies before the origin of thylakoids.

Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

An integrative approach unveils a distal encounter site for rPTPε and phospho-Src complex formation.

The structure determination of protein tyrosine phosphatase (PTP): phospho-protein complexes, which is essential to understand how specificity is achieved at the amino acid level, remains a significant challenge for protein crystallography and cryoEM due to the transient nature of binding interactions. Using rPTPεD1 and phospho-SrcKD as a model system, we have established an integrative workflow to address this problem, by means of which we generate a protein:phospho-protein complex model using predetermined protein structures, SAXS and pTyr-tailored MD simulations. Our model reveals transient protein-protein interactions between rPTPεD1 and phospho-SrcKD and is supported by three independent experimental validations. Measurements of the association rate between rPTPεD1 and phospho-SrcKD showed that mutations on the rPTPεD1: SrcKD complex interface disrupts these transient interactions, resulting in a reduction in protein-protein association rate and, eventually, phosphatase activity. This integrative approach is applicable to other PTP: phospho-protein complexes and the characterization of transient protein-protein interface interactions.

An ATP-sensitive phosphoketolase regulates carbon fixation in cyanobacteria.

Regulation of CO2 fixation in cyanobacteria is important both for the organism and global carbon balance. Here we show that phosphoketolase in Synechococcus elongatus PCC7942 (SeXPK) possesses a distinct ATP-sensing mechanism, where a drop in ATP level allows SeXPK to divert precursors of the RuBisCO substrate away from the Calvin-Benson-Bassham cycle. Deleting the SeXPK gene increased CO2 fixation particularly during light-dark transitions. In high-density cultures, the Δxpk strain showed a 60% increase in carbon fixation and unexpectedly resulted in sucrose secretion without any pathway engineering. Using cryo-EM analysis, we discovered that these functions were enabled by a unique allosteric regulatory site involving two subunits jointly binding two ATP, which constantly suppresses the activity of SeXPK until the ATP level drops. This magnesium-independent ATP allosteric site is present in many species across all three domains of life, where it may also play important regulatory functions.

A domain knowledge enhanced yield based deep learning classifier identifies perineural invasion in oral cavity squamous cell carcinoma.

Background: Perineural invasion (PNI), a form of local invasion defined as the ability of cancer cells to invade in, around, and through nerves, has a negative prognostic impact in oral cavity squamous cell carcinoma (OCSCC). Unfortunately, the diagnosis of PNI suffers from a significant degree of intra- and interobserver variability. The aim of this pilot study was to develop a deep learning-based human-enhanced tool, termed domain knowledge enhanced yield (Domain-KEY) algorithm, for identifying PNI in digital slides. Methods: Hematoxylin and eosin (H&E)-stained whole-slide images (WSIs, n = 85) were obtained from 80 patients with OCSCC. The model structure consisted of two parts to simulate human decision-making skills in diagnostic pathology. To this aim, two semantic segmentation models were constructed (i.e., identification of nerve fibers followed by the diagnosis of PNI). The inferred results were subsequently subjected to post-processing of generated decision rules for diagnostic labeling. Ten H&E-stained WSIs not previously used in the study were read and labeled by the Domain-KEY algorithm. Thereafter, labeling correctness was visually inspected by two independent pathologists. Results: The Domain-KEY algorithm was found to outperform the ResnetV2_50 classifier for the detection of PNI (diagnostic accuracy: 89.01% and 61.94%, respectively). On analyzing WSIs, the algorithm achieved a mean diagnostic accuracy as high as 97.50% versus traditional pathology. The observed accuracy in a validation dataset of 25 WSIs obtained from seven patients with oropharyngeal (cancer of the tongue base, n = 1; tonsil cancer, n = 1; soft palate cancer, n = 1) and hypopharyngeal (cancer of posterior wall, n = 2; pyriform sinus cancer, n = 2) malignancies was 96%. Notably, the algorithm was successfully applied in the analysis of WSIs to shorten the time required to reach a diagnosis. The addition of the hybrid intelligence model decreased the mean time required to reach a diagnosis by 15.0% and 23.7% for the first and second pathologists, respectively. On analyzing digital slides, the tool was effective in supporting human diagnostic thinking. Conclusions: The Domain-KEY algorithm successfully mimicked human decision-making skills and supported expert pathologists in the routine diagnosis of PNI.

Primary intracranial malignant melanomas in solitary type: a tertiary center experience.

PURPOSE: Solitary type primary intracranial malignant melanoma (PIMM) is extremely rare but fatal. The optimal treatment algorithm according to clinical relevance of symptoms and outcomes is unclear. This series emphasized the prognostic factors of solitary PIMM and established the treatment algorithm for this rare disease. METHODS: Patients with solitary PIMMs were pathologically verified and treated with neurosurgical tumor resection. All solitary PIMMs recruited at our institute received multidisciplinary team care. We analyzed the clinical findings and prognostic factors. RESULTS: The study cohort included 10 patients. PIMMs in solitary type impacted middle-aged populations with male predominance in Taiwan. Most patients (80%) presented a single tumor initially. Six patients had progressed to multiplicity after the initial treatment. Rates of tumor bleeding and leptomeningeal metastasis seeding (LS) are high in solitary PIMMs. Patients who had gross-total resection (GTR) had better survival than those who had incomplete resection, with median overall survival (OS) rates of 170.4 months vs. 5.23 months (p = 0.004). Multiplicity, eloquent area involvement, initial tumor bleeding, LS, hydrocephalus, and Karnofsky Performance Score < 80 at diagnosis were associated with negative outcomes in progression-free survival and OS. Adjuvant radiotherapy for patients who had LS and for those who cannot undergo grossly total tumor removal resulted in a good outcome. CONCLUSIONS: GTR demonstrated better outcomes for solitary PIMM. For recurrent tumors, aggressively repeated surgical resection remained beneficial for selected cases. Adjuvant radiotherapy was a treatment option for LS following operation. We proposed a possible treatment algorithm for solitary PIMM.

Serial crystallography captures dynamic control of sequential electron and proton transfer events in a flavoenzyme.

Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD•- isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge allows proton transfer from arginine to FAD•-. Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.

IFIT2-depleted metastatic oral squamous cell carcinoma cells induce muscle atrophy and cancer cachexia in mice.

BACKGROUND: Interferon-induced protein with tetratricopeptide repeat 2 (IFIT2) is a reported metastasis suppressor in oral squamous cell carcinoma (OSCC). Metastases and cachexia may coexist. The effect of cancer metastasis on cancer cachexia is largely unknown. We aimed to address this gap in knowledge by characterizing the cachectic phenotype of an IFIT2-depleted metastatic OSCC mouse model. METHODS: Genetically engineered and xenograft tumour models were used to explore the effect of IFIT2-depleted metastatic OSCC on cancer cachexia. Muscle and organ weight changes, tumour burden, inflammatory cytokine profiles, body composition, food intake, serum albumin and C-reactive protein (CRP) levels, and survival were assessed. The activation of the IL6/p38 pathway in atrophied muscle was measured. RESULTS: IFIT2-depleted metastatic tumours caused marked body weight loss (-18.2% vs. initial body weight, P < 0.001) and a poor survival rate (P < 0.01). Skeletal muscles were markedly smaller in IFIT2-depleted metastatic tumour-bearing mice (quadriceps: -28.7%, gastrocnemius: -29.4%, and tibialis: -24.3%, all P < 0.001). Tumour-derived circulating granulocyte-macrophage colony-stimulating factor (+772.2-fold, P < 0.05), GROα (+1283.7-fold, P < 0.05), IL6 (+245.8-fold, P < 0.001), IL8 (+616.9-fold, P < 0.001), IL18 (+24-fold, P < 0.05), IP10 (+18.8-fold, P < 0.001), CCL2 (+439.2-fold, P < 0.001), CCL22 (+9.1-fold, P < 0.01) and tumour necrosis factor α (+196.8-fold, P < 0.05) were elevated in IFIT2-depleted metastatic tumour-bearing mice. Murine granulocyte colony-stimulating factor (+61.4-fold, P < 0.001) and IL6 (+110.9-fold, P < 0.01) levels were significantly increased in IFIT2-depleted metastatic tumour-bearing mice. Serum CRP level (+82.1%, P < 0.05) was significantly increased in cachectic shIFIT2 mice. Serum albumin level (-26.7%, P < 0.01) was significantly decreased in cachectic shIFIT2 mice. An assessment of body composition revealed decreased fat (-81%, P < 0.001) and lean tissue (-21.7%, P < 0.01), which was consistent with the reduced food intake (-19.3%, P < 0.05). Muscle loss was accompanied by a smaller muscle cross-sectional area (-23.3%, P < 0.05). Muscle atrophy of cachectic IFIT2-depleted metastatic tumour-bearing mice (i.v.-shIFIT2 group) was associated with elevated IL6 (+2.7-fold, P < 0.05), phospho-p38 (+2.8-fold, P < 0.05), and atrogin-1 levels (+2.3-fold, P < 0.05) in the skeletal muscle. Neutralization of IL6 rescued shIFIT2 conditioned medium-induced myotube atrophy (+24.6%, P < 0.01). CONCLUSIONS: Our results suggest that the development of shIFIT2 metastatic OSCC lesions promotes IL6 production and is accompanied by the loss of fat and lean tissue, anorexia, and muscle atrophy. This model is appropriate for the study of OSCC cachexia, especially in linking metastasis with cachexia.

NOTCH1 mutations as prognostic marker in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity with poor prognosis. The dysregulation of Notch signaling pathway has been implicated in the OSCC tumorigenesis. However, the clinical implication of NOTCH1 mutation status in OSCC remains unelucidated. We extracted the NOTCH1 gene mutations from a whole exome sequencing dataset of 168 frozen OSCC tumor specimens and validated these NOTCH1 gene mutations by Sanger sequencing. We also assessed these NOTCH1 gene mutations and its pathological significance in our OSCC tumor tissues using immunohistochemistry. Univariate and multivariate analyses were also used to determine whether the association between NOTCH1 mutation status and prognostic factors was independent of other parameters. In this study, we have identified 44 (26.19 %) NOTCH1 gene mutations from a whole-exome sequencing of 168 OSCC formalin-fixed, paraffin-embedded (FFPE) tissue specimen. These mutations distributed in different NOTCH1 function domains, including the EGF-like repeats region, negative regulatory region, and Ankyrin repeats region. The immunohistochemical staining analysis revealed that NOTCH1 expression was increased in oral cancer tissues. In addition, of the 43 OSCC tumors with NOTCH1 mutations, we observed that the majority were negative for NOTCH1 intracellular domain 1 (NICD1) staining (76.74 %), and 10 tumors were positive for NICD1 staining (23.26 %). In conclusion, our study suggested that NOTCH1 expression is associated with the progression of OSCC. We also demonstrated that presence of a mutated NOTCH1 gene will help prognostic stratification in OSCC when combined with other clinicopathologic parameters.

The Fibronectin Expression Determines the Distinct Progressions of Malignant Gliomas via Transforming Growth Factor-Beta Pathway.

Due to the increasing incidence of malignant gliomas, particularly glioblastoma multiforme (GBM), a simple and reliable GBM diagnosis is needed to screen early the death-threaten patients. This study aimed to identify a protein that can be used to discriminate GBM from low-grade astrocytoma and elucidate further that it has a functional role during malignant glioma progressions. To identify proteins that display low or no expression in low-grade astrocytoma but elevated levels in GBM, glycoprotein fibronectin (FN) was particularly examined according to the mining of the Human Protein Atlas. Web-based open megadata minings revealed that FN was mainly mutated in the cBio Cancer Genomic Portal but dominantly overexpressed in the ONCOMINE (a cancer microarray database and integrated data-mining platform) in distinct tumor types. Furthermore, numerous different cancer patients with high FN indeed exhibited a poor prognosis in the PrognoScan mining, indicating that FN involves in tumor malignancy. To investigate further the significance of FN expression in glioma progression, tumor specimens from five malignant gliomas with recurrences that received at least two surgeries were enrolled and examined. The immunohistochemical staining showed that FN expression indeed determined the distinct progressions of malignant gliomas. Furthermore, the expression of vimentin (VIM), a mesenchymal protein that is strongly expressed in malignant cancers, was similar to the FN pattern. Moreover, the level of epithelial-mesenchymal transition (EMT) inducer transforming growth factor-beta (TGF-ß) was almost recapitulated with the FN expression. Together, this study identifies a protein FN that can be used to diagnose GBM from low-grade astrocytoma; moreover, its expression functionally determines the malignant glioma progressions via TGF-ß-induced EMT pathway.

An anatomical perspective on clinicopathological characteristics and treatment outcomes of dorsal and ventrolateral tongue leukoplakia after carbon dioxide laser surgery.

BACKGROUND: The tongue has been identified as a high-risk site for malignant transformation of oral leukoplakia. The purpose of this study was to investigate the clinicopathological characteristics and treatment outcomes of the dorsal and ventrolateral tongue leukoplakia. METHODS: Demographic data and pathological results of patients who underwent carbon dioxide laser surgery for tongue leukoplakia from 2002 to 2019 were retrospectively reviewed and analyzed statistically. RESULTS: Of the 111 patients enrolled, 80 were males and 31 females, with a mean age of 51.86 ± 11.84 years. The follow-up time was 3.74 ± 4.19 years. Fifteen patients had a postoperative recurrence (13.51%). Four (3.6%) patients developed malignant transformation. Annual transformation rate was 4.03%. There were no differences in the time to develop carcinoma (3.19 ± 1.94 vs. 3.51 ± 2.12 years, P = 0.83), overall cumulative malignant transformation rates (7.41% vs. 2.25%, P = 0.12), and annual transformation rates (2.32% vs. 0.64%, P = 0.099). The prevalence of the ventrolateral tongue leukoplakia was higher than that of the dorsal tongue leukoplakia (P < 0.001). The results of multivariate logistic regression analysis showed that the degree of pathology was the only independent prognostic factor related to postoperative malignant transformation (P = 0.045). CONCLUSIONS: Dorsal tongue leukoplakia is not as frequently encountered clinically as ventrolateral tongue leukoplakia. The response of the dorsal tongue and ventrolateral tongue leukoplakia to laser therapy of are comparable in postoperative recurrence and postoperative malignant transformation. Clinicians should take a more aggressive attitude toward oral tongue leukoplakia with higher grade of dysplasia.