tbuckley/cabot-search huggingface dataset:
B cell tolerance and autoimmunity: Lessons from repertoires
Authors: Jacques Deguine, Ramnik J. Xavier
Journal: The Journal of Experimental Medicine (2024)

Adaptive immune cell function is regulated by a highly diverse receptor recombined from variable germline-encoded segments that can recognize an almost unlimited array of epitopes. While this diversity enables the recognition of any pathogen, it also poses a risk of self-recognition, leading to autoimmunity. Many layers of regulation are present during both the generation and activation of B cells to prevent this phenomenon, although they are evidently imperfect. In recent years, our ability to analyze immune repertoires at scale has drastically increased, both through advances in sequencing and single-cell analyses. Here, we review the current knowledge on B cell repertoire analyses, focusing on their implication for autoimmunity. These studies demonstrate that a failure of tolerance occurs at multiple independent checkpoints in different autoimmune contexts, particularly during B cell maturation, plasmablast differentiation, and within germinal centers. These failures are marked by distinct repertoire features that may be used to identify disease- or patient-specific therapeutic approaches.
Immature B cell homing shapes human lymphoid tissue structure and function
Authors: Jo Spencer, Chiara Dionisi
Journal: The Journal of Experimental Medicine (2024)

Shortly after the emergence of newly formed human B cells from bone marrow as transitional cells, they diverge along two developmental pathways that can be distinguished by the level of IgM they express and migratory biases. Here, we propose that differential tissue homing of immature B cell subsets contributes to human lymphoid tissue structure and function.
Prediction intervals reveal uncertainty in the effect of metabolic syndrome on surgical mortality: A closer look at heterogeneity
Authors: I-Wen Chen, Wei-Ting Wang, Chien-Ming Lin, Kuo-Chuan Hung
Journal: International Journal of Surgery (2024)

Chen, I-Wen MD; Wang, Wei-Ting BSc; Lin, Chien-Ming MD; Hung, Kuo-Chuan MD Author Information
Protective effect of sevoflurane on myocardial ischemia-reperfusion injury: a systematic review and meta-analysis
Authors: Amirhossein Nasiri-Valikboni, Mohamad Rashid, Amir Azimi, Hamed Zarei, Mahmoud Yousefifard
Journal: International Journal of Surgery (2024)

Myocardial ischemia-reperfusion (I/R) injury significantly impacts recovery in both cardiac and non-cardiac surgeries, potentially leading to severe cardiac dysfunction. Sevoflurane, a volatile anesthetic, is reputed for its protective effects against myocardial I/R injury, although evidence remains inconclusive. This systematic review and meta-analysis aim to clarify the cardioprotective efficacy of sevoflurane.
BMScope: A scoping review to chart the evolving clinical study landscape in brain and leptomeningeal metastasis
Authors: Vinton W T Cheng, Richard Heywood, Rasheed Zakaria, Rebecca Burger, Kieran Zucker, Siddarth Kannan, Muhammad Alifian Remifta Putra, Amanda Fitzpatrick, Gary Doherty, Paul Sanghera, Michael D Jenkinson, Carlo Palmieri
Journal: Neuro-Oncology (2024)

Recent studies have challenged the notion that patients with brain metastasis (BM) or leptomeningeal metastasis (LM) should be excluded from systemic therapy clinical trials. This scoping study summarizes the BM/LM clinical studies published between 2010 and 2023.
ENDOLUMINAL RADIOFREQUENCY ABLATION WITH STENTING VS STENTING ONLY IN PATIENTS WITH MALIGNANT BILIARY OBSTRUCTION: A META-ANALYSIS OF RANDOMISED TRIALS
Authors: Daryl Ramai, Marcello Maida, Eric R. Smith, Yichen Wang, Marco Spadaccini, Melissa Previtera, Saurabh Chandan, Yuting Huang, Salih Tokmak, Peter Bhandari, Jason DuBroff, Christopher Ko, John D. Morris, Giovanni Marasco, Stefano Francesco Crinó, Antonio Facciorusso, Benedetto Mangiavillano, Mouen A Khashab
Journal: Endoscopy (2024)

Abstract Background: Endoluminal radiofrequency ablation (RFA) is a palliative treatment for patients suffering from malignant biliary obstruction (MBO). We aimed to conduct a meta-analysis to evaluate the impact of RFA on stent patency, patient survival, and adverse events. Methods: Major databases were searched through December 2023 for patients who had undergone stenting with or without RFA for extrahepatic MBO. A random-effects model was used for analysis, with results expressed as relative risk ratios (RRs) with 95%CIs. Results: Nine RCTs involving 750 subjects with MBO (374 RFA plus stent vs. 376 stent only) were included. Meta-analysis revealed similar risks of stent patency at 3 months (RR 1.01, 95%CI 0.92–1.11; I 2 = 4%) for RFA plus stenting vs. stent only. Meta-analysis showed improved survival at 6 months (RR 0.84, 95%CI 0.73–0.96; I 2 = 21%; P = 0.01) for RFA plus stenting vs. stent only. Subgroup analysis comparing plastic vs. uncovered metal stents showed that stent patency was unaffected at 3 months (RR 1.06, 95%CI 0.91–1.23; I 2 = 17%). Subgroup analysis showed that patients with cholangiocarcinoma experienced an overall survival benefit with RFA plus stenting vs. stenting alone (P &lt; 0.001); however, stent patency remained unaffected (P = 0.08). An increased incidence of cholecystitis was noted with RFA plus stenting vs. stenting alone (5.1% [95%CI 3.1%–7.8%] vs. 0.3% [95%CI 0.01%–1.5%], respectively). Conclusion: Combining endoluminal RFA and stenting may improve overall survival in patients with MBO. RFA did not significantly impact stent patency.
Atezolizumab and chemotherapy for advanced or recurrent endometrial cancer (AtTEnd): a randomised, double-blind, placebo-controlled, phase 3 trial
Authors: Nicoletta Colombo, Elena Biagioli, Kenichi Harano, Francesca Galli, Emma Hudson, Yoland Antill, Chel Hun Choi, Manuela Rabaglio, Frederic Marmé, Christian Marth, Gabriella Parma, Lorena Fariñas-Madrid, Shin Nishio, Karen Allan, Yeh Chen Lee, Elisa Piovano, Beatriz Pardo, Satoshi Nakagawa, John McQueen, Claudio Zamagni, Luis Manso, Kazuhiro Takehara, Giulia Tasca, Annamaria Ferrero, Germana Tognon, Andrea Alberto Lissoni, Mariacristina Petrella, Maria Elena Laudani, Eliana Rulli, Sara Uggeri, M Pilar Barretina Ginesta, Paolo Zola, Claudia Casanova, Valentina Arcangeli, Lorenzo Antonuzzo, Angiolo Gadducci, Stefania Cosio, Andrew Clamp, Mojca Persic, Ian McNeish, Laura Tookman, Andrés Redondo Sanchez, Chel Hun Choi, Editta Baldini, Innocenza Palaia, Pierluigi Benedetti Panici, Nobutaka Takahashi, Janine Lombard, Antonio Ardizzoia, Alessandra Bologna, Ana Maria Herrero Ibáñez, Antonino Musolino, Raúl Márquez Vázquez, Klaus Pietzner, Elena Braicu, Viola A. Heinzelmann-Schwarz, Melanie Powell, Yoshihito Yokoyama, Sally Baron-Hay, Chiara Abeni, Cristina Martin Lorente, Juan Fernando Cueva, Fabian Trillsch, Florian Heitz, Beyhan Ataseven, Edgar Petru, MartinLeonhard Heubner, Azmat Hassanq Sadozye, Sidharth Dubey, Andrea Tazbirkova, Susan Tiley, Kathryn Chrystal, Sang Wun Kim, Mathias Fehr, Kate Scatchard, Anjana Anand, Alexandra Taylor, Hidemichi Watary, Takayuki Enomoto, Kosuke Yoshihara, Sudarsha Selva-Nayagam, Bhaskar Karki, Michelle Harrison, Kate Wilkinson, Jeffrey Goh, Amanda Glasgow, Lorraine Chantrill, Chulmin Lee, Alessandro Bertolini, Filomena Narducci, Giovanna Bellotti, Vittorio Fusco, Stefan Aebi, Maria Del Grande, Ilaria Colombo, Hideki Tokunaga, Shogo Shigeta, Geraldine Goss, Zhen Rong Siow, Christopher Steer, Hao Lin, Kwang-Beom Lee, Giovanni Di Meglio, Elena Massa, Elvira De Marino, Vincenzo Tortora, Isabel Palacio Vazquez, Kosuke Tsuji, Eiichiro Tominaga, Jeffrey Goh, Allison Black, Kyeong A So, Dong Hoon Suh, Keun Ho Lee, Yong Man Kim, Roldano Fossati, Luciano Carlucci, Massimo Barberis, Valter Torri, Anna Santoni
Journal: The Lancet Oncology (2024)
Identifying risk factors for anal cancer in people with HIV in Spain: a multicentre retrospective cohort study nested in the PISCIS cohort
Authors: Josep M Llibre, Boris Revollo, Jordi Aceiton, Yesika Díaz, Pere Domingo, Joaquim Burgos, Patricia Sorni, Maria Saumoy, Hernando Knobel, Marta Navarro, Elena Leon, Amat Orti, Laia Arbonés, Arantxa Mera, Elisabet Deig, Guillem Sirera, Josep M Miró, Jordi Casabona, Raquel Martin-Iguacel
Journal: The Lancet HIV (2024)
A brain cell atlas integrating single-cell transcriptomes across human brain regions
Authors: Xinyue Chen, Yin Huang, Liangfeng Huang, Ziliang Huang, Zhao-Zhe Hao, Lahong Xu, Nana Xu, Zhi Li, Yonggao Mou, Mingli Ye, Renke You, Xuegong Zhang, Sheng Liu, Zhichao Miao
Journal: Nature Medicine (2024)

While single-cell technologies have greatly advanced our comprehension of human brain cell types and functions, studies including large numbers of donors and multiple brain regions are needed to extend our understanding of brain cell heterogeneity. Integrating atlas-level single-cell data presents a chance to reveal rare cell types and cellular heterogeneity across brain regions. Here we present the Brain Cell Atlas, a comprehensive reference atlas of brain cells, by assembling single-cell data from 70 human and 103 mouse studies of the brain throughout major developmental stages across brain regions, covering over 26.3 million cells or nuclei from both healthy and diseased tissues. Using machine-learning based algorithms, the Brain Cell Atlas provides a consensus cell type annotation, and it showcases the identification of putative neural progenitor cells and a cell subpopulation of PCDH9
Evolutionary changes in cognition due to fisheries mortality?
Authors: Tamal Roy, Alexander Kotrschal, Robert Arlinghaus
Journal: Trends in Ecology & Evolution (2024)

Fish experiencing harvest mortality often evolve a fast life-history that prioritizes investment in current versus future reproduction, thereby potentially limiting energetic investment in the brain. Fisheries may also select for shy fish that are less willing to learn, or directly select fish with poor cognitive ability. The resulting evolutionary changes can alter the cognitive performance of individuals and affect fish populations and fisheries quality.
Sex-based Differences in Risk for Therapy-related Myeloid Neoplasms
Authors: Melissa A. Richard, Chengcheng Yan, Yanjun Chen, Christopher J. Gibson, Rashi Kalra, Alysia Bosworth, David K. Crossman, Purnima Singh, Lindsey Hageman, Jianbo He, Saro H. Armenian, Julie Vose, Daniel J. Weisdorf, Benjamin L Ebert, Yutaka Yasui, Changde Cheng, Stephen J. Forman, Smita Bhatia, Ravi Bhatia
Journal: Journal of Clinical Oncology (2024)

PURPOSE Therapy-related myeloid neoplasm (t-MN) is a life-threatening complication of autologous peripheral blood stem cell (PBSC) transplantation for non-Hodgkin lymphoma (NHL). Previous studies report an association between clonal hematopoiesis (CH) in PBSC and risk of t-MN, but small samples precluded examination of risk within specific subpopulations. METHODS Targeted DNA sequencing was performed to identify CH mutations in PBSC from a retrospective cohort of 984 patients with NHL (median age at transplant, 57 years; range, 18-78). Fine-Gray proportional subdistribution hazard regression models estimated association between number of CH mutations and t-MN, adjusting for demographic, clinical, and therapeutic variables. Secondary analyses evaluated the association between CH and t-MN among males and females. RESULTS CH was identified in PBSC from 366 patients (37.2%). t-MN developed in 60 patients after a median follow-up of 5 years. Presence of ≥2 mutations conferred increased t-MN risk (adjusted hazard ratio [aHR], 2.10; 95% CI [1.08 to 4.11]; P = .029). CH was associated with increased t-MN risk among males (aHR, 1.83 [95% CI, 1.01 to 3.31]) but not females (aHR, 0.56 [95% CI, 0.15 to 2.09]). Although the prevalence and type of CH mutations in PBSC were comparable, the 8-year cumulative incidence of t-MN was higher among males vs. females with CH (12.4% v 3.6%) but was similar between males and females without CH (4.9% v 3.9%). Expansion of CH clones from PBSC to t-MN was seen only among males. CONCLUSION presence of CH mutations in PBSC confers increased risk of t-MN after autologous transplantation in male but not female patients with NHL. Factors underlying sex-based differences in risk of CH progression to t-MN merit further investigation.
Overcoming Systemic Barriers to Make Patient-Partnered Research a Reality
Authors: Vinayak Venkataraman, Bailey A. Martin-Giacalone, Bettina F. Drake, Liz Salmi, Elizabeth B. Claus, Anne L.R. Schuster, John F.P. Bridges, Heinz-Josef Lenz, Cheryl L. Willman, Diane Diehl, Katherine A. Janeway, Jennifer W. Mack, Suzanne George
Journal: Journal of Clinical Oncology (2024)
Durability of Response With Selpercatinib in Patients With <i>RET</i>-Activated Thyroid Cancer: Long-Term Safety and Efficacy From LIBRETTO-001
Authors: Lori J. Wirth, Marcia S. Brose, Vivek Subbiah, Francis Worden, Ben Solomon, Bruce Robinson, Julien Hadoux, Pascale Tomasini, Daniela Weiler, Barbara Deschler-Baier, Daniel S.W. Tan, Patricia Maeda, Yan Lin, Ravinder Singh, Theresa Bayt, Alexander Drilon, Philippe A. Cassier
Journal: Journal of Clinical Oncology (2024)

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported. LIBRETTO-001 is a registrational phase I/II, single-arm, open-label study of selpercatinib in patients with RET (REarranged during Transfection)-activated cancers (ClinicalTrials.gov identifier: NCT03157128 ). We present long-term safety and efficacy from LIBRETTO-001 in patients with RET-mutant medullary thyroid cancer (MTC; n = 324) and RET fusion-positive thyroid cancer encompassing different histological subtypes (TC; n = 66). At the data cutoff of January 2023, the objective response rate was 82.5% among patients with cabozantinib/vandetanib-naïve MTC and 95.8% among patients with treatment-naïve TC. At a median follow-up time of 42.4 and 44.0 months in patients with cabozantinib/vandetanib-naïve and pretreated MTC, the median progression-free survival (PFS) was not reached and 41.4 months, respectively. At a median follow-up time of 24.9 and 30.4 months in patients with treatment-naïve and pretreated TC, the median PFS was not reached and 27.4 months, respectively. Three-year PFS rates were 75.2% and 87.3% among patients with cabozantinib/vandetanib-naïve MTC and treatment-naïve TC, respectively. Median PFS was similar to median duration of response for each patient group. The safety profile of selpercatinib was consistent with previous reports. With an additional follow-up of 37 months and 228 more patients from the last disclosure, selpercatinib continued to provide durable and robust responses in treatment-naïve and previously treated patients with RET-mutant MTC and RET fusion-positive TC.
Trichloroethylene: An Update on an Environmental Contaminant with Multiple Health Effects
Authors: Lawrence H. Lash
Journal: The Annual Review of Pharmacology and Toxicology (2024)

The halogenated solvent trichloroethylene (TCE) has had many uses in medicine, construction, consumer products, and the military. Many of these uses have been discontinued or restricted due to its toxicity, which affects multiple target organs and includes both acute, high-dose toxicity and chronic, low-dose toxicity that also encompass several cancers. US and international agencies have conducted risk and hazard assessments for TCE, with comprehensive publications coming out in the last 10-15 years. Accordingly, the focus of this article is to review recently published data since that time (i.e., 2014) that clarify unsettled questions or provide additional insights into the metabolism and mechanisms of toxicity of TCE in several target organs. Besides metabolism, the review focuses on the kidneys, liver, immune system, nervous system, cardiovascular and pulmonary systems, the search for biomarkers, and recent analyses of human cancer risk and incidence from TCE exposure.
An Alternatively Spliced Gain-of-Function NT5C2 Isoform Contributes to Chemoresistance in Acute Lymphoblastic Leukemia
Authors: Manuel Torres-Diz, Clara Reglero, Catherine D. Falkenstein, Annette Castro, Katharina E. Hayer, Caleb M. Radens, Mathieu Quesnel-Vallières, Zhiwei Ang, Priyanka Sehgal, Marilyn M. Li, Yoseph Barash, Sarah K. Tasian, Adolfo Ferrando, Andrei Thomas-Tikhonenko
Journal: Cancer Research (2024)

Abstract Relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) is a major cause of pediatric cancer–related deaths. Relapse-specific mutations do not account for all chemotherapy failures in B-ALL patients, suggesting additional mechanisms of resistance. By mining RNA sequencing datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive alternative splicing (AS) patterns linked to relapse and affecting drivers of resistance to glucocorticoids, antifolates, and thiopurines. Most splicing variations represented cassette exon skipping, “poison” exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In contrast, relapse-associated AS of NT5C2 mRNA yielded an isoform with the functionally uncharacterized in-frame exon 6a. Incorporation of the 8-amino acid sequence SQVAVQKR into this enzyme created a putative phosphorylation site and resulted in elevated nucleosidase activity, which is a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-mercaptopurine in B-ALL cells both in vitro and in vivo. Furthermore, both NT5C2ex6a and the R238W variant induced collateral sensitivity to the inosine monophosphate dehydrogenase inhibitor mizoribine. These results ascribe to splicing perturbations an important role in chemotherapy resistance in relapsed B-ALL and suggest that inosine monophosphate dehydrogenase inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias. Significance: Alternative splicing is a potent mechanism of acquired drug resistance in relapsed/refractory acute lymphoblastic leukemias that has diagnostic and therapeutic implications for patients who lack mutations in known chemoresistance genes.
Five-Year Follow-Up of Standard-of-Care Axicabtagene Ciloleucel for Large B-Cell Lymphoma: Results From the US Lymphoma CAR T Consortium
Authors: Michael D. Jain, Jay Y. Spiegel, Loretta J. Nastoupil, John Tamaresis, Armin Ghobadi, Yi Lin, Lazaros Lekakis, Patrick Reagan, Olalekan Oluwole, Joseph McGuirk, Abhinav Deol, Kathleen A. Dorritie, Alison R. Sehgal, Andre Goy, Brian T. Hill, Charalambos Andreadis, Javier Munoz, Matthew Ulrickson, Jason Westin, Julio C. Chavez, Dilan Patel, Miriam T. Jacobs, Radhika Bansal, N. Nora Bennani, Vivek G. Patel, Aaron P. Rapoport, Julie M. Vose, David B. Miklos, Sattva S. Neelapu, Frederick L. Locke, Matthew Lunning, Saurabh Dahiya
Journal: Journal of Clinical Oncology (2024)

Axicabtagene ciloleucel (axi-cel) is an autologous CD19 chimeric antigen receptor (CAR) T-cell therapy that is approved for the treatment of relapsed or refractory large B-cell lymphoma. Little is known about the long-term survivorship after CAR T-cell therapy.
Associations of the EAT-Lancet reference diet with metabolic dysfunction–associated steatotic liver disease and its severity: A multicohort study
Authors: Shunming Zhang, Yan Yan, Xu-Fen Zeng, Yeqing Gu, Hongmei Wu, Qing Zhang, Li Liu, Zhenyu Huo, Xiaoqin Luo, Rui Zhang, Emily Sonestedt, Yan Borné, Lu Qi, Tao Huang, Ming-Hua Zheng, Yu-Ming Chen, Kaijun Niu, Le Ma
Journal: Hepatology (2024)

The EAT-Lancet Commission devised a globally sustainable dietary pattern to jointly promote human health and sustainability. However, the extent to which this diet supports metabolic dysfunction-associated steatotic liver disease (MASLD) has not yet been assessed. This study aimed to investigate the association between the EAT-Lancet diet and the risk of MASLD and its severity.
Reply: Don’t forget to discuss TIPS in patients surviving an episode of alcoholic hepatitis
Authors: Jordi Gratacós-Ginès, Meritxell Ventura-Cots, Elisa Pose
Journal: Hepatology (2024)

We read the letter by Rudler and colleagues in reference to our study on recurrent alcohol-associated hepatitis (AH) and would like to comment on some of the issues raised. Regarding noninvasive diagnostic criteria of AH, it has been consistently shown that their accuracy is not higher than 80%. We recently proposed new criteria,1 which were validated in the study cited by Rudler and colleagues. Nevertheless, performing a liver biopsy in this setting is not standard practice as per clinical practice guidelines. Our study on recurrent AH is a multicenter analysis with a pragmatic design in which most centers did not have transjugular biopsy available. Inclusion criteria were based on NIAAA Consortium recommendations and were consistent with previous studies in the field. With regard to the indication for preemptive TIPS (pTIPS), several comments are needed. First, it should be noted that the applicability of pTIPS in AH is low: in our study, only 23 patients (2% of the cohort and 32% of those bleeding) were candidates according to current criteria. Second, evidence on the efficacy and safety of pTIPS in AH is lacking: previous trials do not provide data on the number of patients with AH enrolled but mean bilirubin levels are well below the diagnostic cutoff for AH, suggesting that very few patients with AH were enrolled.2 Consequently, conclusions on the applicability of pTIPS should be taken with caution when applied to AH. Third, AH has a specific natural history and distinct HVPG dynamics compared to decompensated cirrhosis: following an episode of AH, abstinent patients and those occasionally consuming often achieve a spontaneous reduction in HVPG of more than 20%,3 thus reducing their risk of recurrent bleeding and death.4 In contrast to the weak evidence of pTIPS in AH, many studies have shown that alcohol abstinence is the most important prognostic factor across all stages of alcohol-associated liver disease.5 In our opinion, TIPS should be used with caution in AH until specific information becomes available. Conversely, we believe efforts should be directed toward the treatment of alcohol use disorder.
Reply to Divo and Celli: To Our Knowledge, You Are Not the First…
Authors: Daniel J Tan, E. Haydn Walters, Don Vicendese, Jennifer L. Perret, Shyamali C. Dharmage
Journal: American Journal of Respiratory and Critical Care Medicine (2024)

"Reply to Divo and Celli: To Our Knowledge, You Are Not the First…." American Journal of Respiratory and Critical Care Medicine, 0(ja), pp.
Omalizumab for the treatment of food allergies … and other research
Journal: BMJ (2024)

Omalizumab for the treatment of food allergies … and other researchIn this Opinion by Ann Robinson (BMJ 2024;384:q547; doi:) the first item should have stated: "Apart from scrupulous avoidance of allergens, which is easier said than done, the only approved treatment is oral immunotherapy for peanut allergy" [not "… oral immunotherapy, omalizumab, for peanut allergy"].Omalizumab is an injectable monoclonal anti-IgE antibody used to treat severe persistent allergic forms of asthma, nasal polyps, and urticaria (and in the US since February 2024 food allergy).The study summarised by Ann Robinson had investigated whether omalizumab would be effective and safe as monotherapy in patients with multiple food allergies.
Does the METHOD study show that ZOE works?
Authors: Margaret McCartney, Deborah Cohen
Journal: BMJ (2024)

The evidence is in: ZOE works."The email from ZOE, sent to people who had signed up for information about it, went on "Most health and wellness companies don't test their products scientifically.So, there's no proof that they work.This is where we're different.As a science-first company, we decided to put ZOE to the test with a rigorously conducted