Xylosyltransferase Bump-and-hole Engineering to Chemically Manipulate Proteoglycans in Mammalian Cells.

Mammalian cells orchestrate signalling through interaction events on their surfaces. Proteoglycans are an intricate part of these interactions, carrying large glycosaminoglycan polysaccharides that recruit signalling molecules. Despite their importance in development, cancer and neurobiology, a relatively small number of proteoglycans have been identified. In addition to the complexity of glycan extension, biosynthetic redundancy in the first protein glycosylation step by two xylosyltransferase isoenzymes XT1 and XT2 complicates annotation of proteoglycans. Here, we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. By employing a tactic termed bump- and-hole engineering, we engineer the two isoenzymes XT1 and XT2 to specifically transfer a chemically modified xylose analogue to target proteins. The chemical modification contains a bioorthogonal tag, allowing the ability to visualise and profile target proteins modified by both transferases in mammalian cells. The versatility of our approach allows pinpointing glycosylation sites by tandem mass spectrometry, and exploiting the chemical handle to manufacture proteoglycans with defined GAG chains for cellular applications. Engineered XT enzymes permit a view into proteoglycan biology that is orthogonal to conventional techniques in biochemistry.

Analysis of Mucin-Domain Glycoproteins Using Mass Spectrometry.

Mucin-domain glycoproteins are characterized by their high density of glycosylated serine and threonine residues, which complicates their analysis by mass spectrometry. The dense glycosylation renders the protein backbone inaccessible to workhorse proteases like trypsin, the vast heterogeneity of glycosylation often results in ion suppression from unmodified peptides, and search algorithms struggle to confidently analyze and site-localize O-glycosites. We have made a number of advances to address these challenges, rendering mucinomics possible for the first time. Here, we summarize these contributions and provide a detailed protocol for mass spectrometric analysis of mucin-domain glycoproteins. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Enrichment of mucin-domain glycoproteins Basic Protocol 2: Enzymatic digestion of mucin-domain glycoprotein(s) Basic Protocol 3: Mass spectrometry data collection for O-glycopeptides Basic Protocol 4: Mass spectrometry data analysis of O-glycopeptides.

The thin red line: Blood planning factors and the enduring need for a robust military blood system to support combat operations.

ABSTRACT: Battlefield lessons learned are forgotten; the current name for this is the Walker Dip. Blood transfusion and the need for a Department of Defense Blood Program are lessons that have cycled through being learned during wartime, forgotten, and then relearned during the next war. The military will always need a blood program to support combat and contingency operations. Also, blood supply to the battlefield has planning factors that have been consistent over a century. In 2024, it is imperative that we codify these lessons learned. The linchpins of modern combat casualty care are optimal prehospital care, early whole blood transfusion, and forward surgical care. This current opinion comprised of authors from all three military Services, the Joint Trauma System, the Armed Services Blood Program, blood SMEs and the CCC Research Program discuss two vital necessities for a successful military trauma system: (1) the need for an Armed Services Blood Program and (2) Planning factors for current and future deployed military ere is no effective care for wounded soldiers, and by extension there is no effective military medicine.

PRC1.6 localizes on chromatin with the human silencing hub (HUSH) complex for promoter-specific silencing.

An obligate step in the life cycle of HIV-1 and other retroviruses is the establishment of the provirus in target cell chromosomes. Transcriptional regulation of proviruses is complex, and understanding the mechanisms underlying this regulation has ramifications for fundamental biology, human health, and gene therapy implementation. The three core components of the Human Silencing Hub (HUSH) complex, TASOR, MPHOSPH8 (MPP8), and PPHLN1 (Periphilin 1), were identified in forward genetic screens for host genes that repress provirus expression. Subsequent loss-of-function screens revealed accessory proteins that collaborate with the HUSH complex to silence proviruses in particular contexts. To identify proteins associated with a HUSH complex-repressed provirus in human cells, we developed a technique, Provirus Proximal Proteomics, based on proximity labeling with C-BERST (dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging). Our screen exploited a lentiviral reporter that is silenced by the HUSH complex in a manner that is independent of the integration site in chromatin. Our data reveal that proviruses silenced by the HUSH complex are associated with DNA repair, mRNA processing, and transcriptional silencing proteins, including L3MBTL2, a member of the non-canonical polycomb repressive complex 1.6 (PRC1.6). A forward genetic screen confirmed that PRC1.6 components L3MBTL2 and MGA contribute to HUSH complex-mediated silencing. PRC1.6 was then shown to silence HUSH-sensitive proviruses in a promoter-specific manner. Genome wide profiling showed striking colocalization of the PRC1.6 and HUSH complexes on chromatin, primarily at sites of active promoters. Finally, PRC1.6 binding at a subset of genes that are silenced by the HUSH complex was dependent on the core HUSH complex component MPP8. These studies offer new tools with great potential for studying the transcriptional regulation of proviruses and reveal crosstalk between the HUSH complex and PRC1.6.

Quantification and site-specific analysis of co-occupied N- and O-glycopeptides.

Protein glycosylation is a complex post-translational modification that is generally classified as N- or O-linked. Site-specific analysis of glycopeptides is accomplished with a variety of fragmentation methods, depending on the type of glycosylation being investigated and the instrumentation available. For instance, collisional dissociation methods are frequently used for N-glycoproteomic analysis with the assumption that one N-sequon exists per tryptic peptide. Alternatively, electron-based methods are indispensable for O-glycosite localization. However, the presence of simultaneously N- and O-glycosylated peptides could suggest the necessity of electron-based fragmentation methods for N-glycoproteomics, which is not commonly performed. Thus, we quantified the prevalence of N- and O-glycopeptides in mucins and other glycoproteins. A much higher frequency of co-occupancy within mucins was detected whereas only a negligible occurrence occurred within non-mucin glycoproteins. This was demonstrated from analyses of recombinant and/or purified proteins, as well as more complex samples. Where co-occupancy occurred, O-glycosites were frequently localized to the Ser/Thr within the N-sequon. Additionally, we found that O-glycans in close proximity to the occupied Asn were predominantly unelaborated core 1 structures, while those further away were more extended. Overall, we demonstrate electron-based methods are required for robust site-specific analysis of mucins, wherein co-occupancy is more prevalent. Conversely, collisional methods are generally sufficient for analyses of other types of glycoproteins.

Noninvasive Ventilation for Preoxygenation during Emergency Intubation.

BACKGROUND: Among critically ill adults undergoing tracheal intubation, hypoxemia increases the risk of cardiac arrest and death. The effect of preoxygenation with noninvasive ventilation, as compared with preoxygenation with an oxygen mask, on the incidence of hypoxemia during tracheal intubation is uncertain. METHODS: In a multicenter, randomized trial conducted at 24 emergency departments and intensive care units in the United States, we randomly assigned critically ill adults (age, ≥18 years) undergoing tracheal intubation to receive preoxygenation with either noninvasive ventilation or an oxygen mask. The primary outcome was hypoxemia during intubation, defined by an oxygen saturation of less than 85% during the interval between induction of anesthesia and 2 minutes after tracheal intubation. RESULTS: Among the 1301 patients enrolled, hypoxemia occurred in 57 of 624 patients (9.1%) in the noninvasive-ventilation group and in 118 of 637 patients (18.5%) in the oxygen-mask group (difference, -9.4 percentage points; 95% confidence interval [CI], -13.2 to -5.6; P<0.001). Cardiac arrest occurred in 1 patient (0.2%) in the noninvasive-ventilation group and in 7 patients (1.1%) in the oxygen-mask group (difference, -0.9 percentage points; 95% CI, -1.8 to -0.1). Aspiration occurred in 6 patients (0.9%) in the noninvasive-ventilation group and in 9 patients (1.4%) in the oxygen-mask group (difference, -0.4 percentage points; 95% CI, -1.6 to 0.7). CONCLUSIONS: Among critically ill adults undergoing tracheal intubation, preoxygenation with noninvasive ventilation resulted in a lower incidence of hypoxemia during intubation than preoxygenation with an oxygen mask. (Funded by the U.S. Department of Defense; PREOXI ClinicalTrials.gov number, NCT05267652.).

Renoprotective Effects of Metabolic Surgery Versus GLP1 Receptor Agonists on Progression of Kidney Impairment in Patients with Established Kidney Disease.

OBJECTIVE: To examine the renoprotective effects of metabolic surgery in patients with established chronic kidney disease (CKD). BACKGROUND: The impact of metabolic surgery compared with glucagon-like peptide-1 receptor agonists (GLP-1RA) in patients with established CKD has not been fully characterized. METHODS: Patients with obesity (BMI ≥30 kg/m2), type 2 diabetes (T2DM), and baseline estimated glomerular filtration rate (eGFR) 20-60 mL/min/1.73 m² who underwent metabolic bariatric surgery at a large U.S. health system (2010-2017) were compared with nonsurgical patients who continuously received GLP-1RA. The primary end point was CKD progression, defined as decline of eGFR by ≥50% or to <15 mL/min/1.73 m2, initiation of dialysis, or kidney transplant. The secondary end point was the incident kidney failure (eGFR <15 mL/min/1.73 m2, dialysis, or kidney transplant) or all-cause mortality. RESULTS: 425 patients, including 183 patients in the metabolic surgery group and 242 patients in the GLP-1RA group, with a median follow-up of 5.8 years (IQR, 4.4-7.6) were analyzed. The cumulative incidence of the primary end point at 8-years was 21.7% (95% CI, 12.2-30.6) in the surgical group and 45.1% (95% CI, 27.7-58.4) in the nonsurgical group, with an adjusted hazard ratio of 0.40 (95% CI, 0.21-0.76), P=0.006. The cumulative incidence of the secondary composite end point at 8-years was 24.0% (95% CI, 14.1-33.2) in the surgical group and 43.8% (95% CI, 28.1-56.1) in the nonsurgical group, with an adjusted HR of 0.56 (95% CI, 0.31-0.99), P=0.048. CONCLUSIONS: Among patients with T2DM, obesity, and established CKD, metabolic surgery, compared with GLP-1RA, was significantly associated with a 60% lower risk of progression of kidney impairment and a 44% lower risk of kidney failure or death. Metabolic surgery should be considered as a therapeutic option for patients with CKD and obesity.

Advancing Combat Casualty Care Statistics and Other Battlefield Care Metrics.

Aggregate statistics can provide intra-conflict and inter-conflict mortality comparisons and trends within and between U.S. combat operations. However, capturing individual-level data to evaluate medical and non-medical factors that influence combat casualty mortality has historically proven difficult. The Department of Defense (DoD) Trauma Registry, developed as an integral component of the Joint Trauma System during recent conflicts in Afghanistan and Iraq, has amassed individual-level data that have afforded greater opportunity for a variety of analyses and comparisons. Although aggregate statistics are easily calculated and commonly used across the DoD, other issues that require consideration include the impact of individual medical interventions, non-medical factors, non-battle-injured casualties, and incomplete or missing medical data, especially for prehospital care and forward surgical team care. Needed are novel methods to address these issues in order to provide a clearer interpretation of aggregate statistics and to highlight solutions that will ultimately increase survival and eliminate preventable death on the battlefield. Although many U.S. military combat fatalities sustain injuries deemed non-survivable, survival among these casualties might be improved using primary and secondary prevention strategies that prevent injury or reduce injury severity. The current commentary proposes adjustments to traditional aggregate combat casualty care statistics by integrating statistics from the DoD Military Trauma Mortality Review process as conducted by the Joint Trauma System and Armed Forces Medical Examiner System.

Feasibility and acceptability of general practitioners using sit-stand desks: a feasibility trial.

OBJECTIVES: The primary aim of this study was to investigate the feasibility and acceptability of general practitioners (GPs) using sit-stand desks to facilitate standing during consultations. A further aim was to examine the views of patients about GPs standing for their consultations. DESIGN: A pre-post single-group experimental trial design. SETTING: General practices in England, UK. PARTICIPANTS: 42 GPs (working a minimum of five clinical sessions per week) and 301 patients (aged ≥18 years). INTERVENTIONS: The intervention consisted of each GP having a sit-stand desk (Opløft Sit-Stand Platform) installed in their consultation room for 4 working weeks. Sit-stand desks allow users to switch, in a few seconds, between a sitting and standing position and vice versa, by adjusting the height of the desk. MAIN OUTCOME MEASURES: To test feasibility and acceptability, GPs reported their views about using sit-stand desks at work at baseline and follow-up. Sitting time and physical activity were also measured via accelerometer at baseline and follow-up. Patients who attended a consultation where their GP was standing were asked to complete an exit questionnaire about the perceived impact on the consultation. RESULTS: Most GPs reported using their sit-stand desk daily (n=28, 75.7%). 16 GPs (44.4%) used their sit-stand desk during face-to-face consultations every day. Most GPs and patients did not view that GPs standing during face-to-face consultations impacted the doctor-patient relationship (GPs; 73.5%, patients; 83.7%). GPs' sitting time during work was 121 min per day lower (95% CI: -165 to -77.58) at follow-up compared with baseline. CONCLUSIONS: Use of sit-stand desks is acceptable within general practice and may reduce sitting time in GPs. This may benefit GPs and help reduce sitting time in patients. TRIAL REGISTRATION NUMBER: ISRCTN76982860.

Developing supervisory and precepting skills through simulation: Near peer teaching in a skills laboratory.

BACKGROUND AND PURPOSE: Near-peer teaching is an innovative approach to teaching the skills of supervising and precepting while benefiting students with different levels of experience and academic training. This study describes near-peer activities in skills-based laboratory courses that provided opportunities for one-on-one teaching to benefit learners in the introductory lab courses while simultaneously training more advanced students for future supervisory and precepting roles. EDUCATIONAL ACTIVITY & SETTING: Three community pharmacy near-peer teaching simulations were designed and implemented: 1) Patient Counseling and Medication Adherence, 2) Dispensing and Patient Counseling, and 3) Drug Utilization Review and Prescriber Calls. These activities took place over two semesters of a pharmacy skills lab with all first- and third-year Doctor of Pharmacy students. FINDINGS: In Autumn 2019, 80% (111/139) of P1s and 67% (80/119) of P3s responded to the course evaluation survey. In Spring 2020, 73% (100/137) of P1s and 68% (80/118) of P3s responded to the course evaluation survey. The P3s reported increased confidence in their ability to provide meaningful feedback, while P1s reported increased confidence in communicating with patients and healthcare providers. Performance data revealed that most P1s and P3s completed dispensing and communication activities accurately using a near-peer approach. Overall, the P1s and P3s felt the activities were valuable learning experiences. SUMMARY: The near-peer activities described in this study fill a gap in the training of pharmacy graduates for future precepting and supervisory roles. Evaluation of these near-peer activities suggest that both junior and senior learners benefit from simulated preceptor-intern interactions, supporting this innovative approach to address supervisory and precepting responsibilities.

Clinical Outcomes of Orthopedic Surgery Comanagement by Internal Medicine Advanced Practice Clinicians: A Cohort Study.

OBJECTIVE: Compare clinical outcomes between advanced practice clinician-based comanagement and usual orthopedic care. BACKGROUND: Comanagement of orthopedic surgery patients by internal medicine hospitalists is associated with improvements in clinical outcomes including complications, length of stay, and cost. Clinical outcomes of orthopedic comanagement performed solely by internal medicine advanced practice clinicians have not been reported. METHODS: Retrospective cohort study in patients 18 years or older, hospitalized for orthopedic joint or spine surgery between May 1, 2014, and January 1, 2022. Outcomes assessed were length of stay, ICU transfer, return to operating room, inhospital and 30-day mortality, 30-day readmission, and total direct cost excluding surgical implants. Generalized boosted regression and propensity score weighting was used to compare clinical outcomes and healthcare cost between usual care and advanced practice clinician comanagement. RESULTS: Advanced practice clinician comanagement was associated with a 5% reduction in mean length of stay (RR=0.95, p=0.009), decreased odds of returning to the operating room (OR= 0.51, p=0.002), and a significant reduction in 30-day mortality (OR= 0.32, p=0.037) compared to usual orthopedic care in a weighted analysis. Need for ICU transfer was higher with advanced practice clinician comanagement (OR= 1.54, p=0.009), without significant differences in 30-day readmission or inhospital mortality. CONCLUSIONS: We observed reductions in length of stay, healthcare costs, return to the operating room, and 30-day mortality with advanced practice clinician comanagement compared to usual orthopedic care. Our findings suggest advanced practice clinician-based comanagement may represent a safe and cost-effective model for orthopedic comanagement.

Obesity, Challenges, and Weight-Loss Strategies for Patients With Ventricular Assist Devices.

For adults with advanced heart failure, class II/III obesity (body mass index ≥35 kg/m2) represents major challenges, and it is even considered a contraindication for heart transplantation (HT) at many centers. This has led to growing interest in preventing and treating obesity to help patients with advanced heart failure become HT candidates. Among all weight-loss strategies, bariatric surgery (BSx) has the greatest weight loss efficacy and has shown value in enabling select patients with left ventricular assist devices (LVADs) and obesity to lose sufficient weight to access HT. Nevertheless, both BSx and antiobesity medications warrant caution in the LVAD population. In this review, the authors describe and interpret the available published reports on the impact of obesity and weight-loss strategies for patients with LVADs from general and HT candidacy standpoints. The authors also provide an overview of the journey of LVAD recipients who undergo BSx and review major aspects of perioperative protocols.

Variation in completeness of coding external cause of injuries under ICD-10-CM.

INTRODUCTION: Information about causes of injury is key for injury prevention efforts. Historically, cause-of-injury coding in clinical practice has been incomplete due to the need for extra diagnosis codes in the International Classification of Diseases-Ninth Revision-Clinical Modification (ICD-9-CM) coding. The transition to ICD-10-CM and increased use of clinical support software for diagnosis coding is expected to improve completeness of cause-of-injury coding. This paper assesses the recording of external cause-of-injury codes specifically for those diagnoses where an additional code is still required. METHODS: We used electronic health record and claims data from 10 health systems from October 2015 to December 2021 to identify all inpatient and emergency encounters with a primary diagnosis of injury. The proportion of encounters that also included a valid external cause-of-injury code is presented. RESULTS: Most health systems had high rates of cause-of-injury coding: over 85% in emergency departments and over 75% in inpatient encounters with primary injury diagnoses. However, several sites had lower rates in both settings. State mandates were associated with consistently high external cause recording. CONCLUSIONS: Completeness of cause-of-injury coding improved since the adoption of ICD-10-CM coding and increased slightly over the study period at most sites. However, significant variation remained, and completeness of cause-of-injury coding in any diagnosis data used for injury prevention planning should be empirically determined.

Nursing science at Mayo Clinic: An alternative model to traditional hospital-based nurse scientist positions.

The role of the Nurse Scientist in clinical settings represents a relatively new career path that has garnered attention in recent literature. Although there is considerable variability in how this role is operationalized across institutions, Mayo Clinic stands out as one of the few health systems in the United States employing nurse scientists who are fully and exclusively engaged in their own programs of research. Given the need for practical information to guide development and implementation of a research-focused nurse scientist role, the purpose of this paper is to describe the infrastructure and resources supporting Mayo Clinic nurse scientists, share role expectations and metrics for success, discuss both the facilitators of success and ongoing challenges, and compare our current practices to those found in the literature.

Diagnosis and Management of Fetal Arrhythmias in the Current Era.

Diagnosis and management of fetal arrhythmias have changed over the past 40-50 years since propranolol was first used to treat fetal tachycardia in 1975 and when first attempts were made at in utero pacing for complete heart block in 1986. Ongoing clinical trials, including the FAST therapy trial for fetal tachycardia and the STOP-BLOQ trial for anti-Ro-mediated fetal heart block, are working to improve diagnosis and management of fetal arrhythmias for both mother and fetus. We are also learning more about how "silent arrhythmias", like long QT syndrome and other inherited channelopathies, may be identified by recognizing "subtle" abnormalities in fetal heart rate, and while echocardiography yet remains the primary tool for diagnosing fetal arrhythmias, research efforts continue to advance the clinical envelope for fetal electrocardiography and fetal magnetocardiography. Pharmacologic management of fetal arrhythmias remains one of the most successful achievements of fetal intervention. Patience, vigilance, and multidisciplinary collaboration are key to successful diagnosis and treatment.

Clinician perspectives on adolescent cannabis-related beliefs and behaviors following recreational cannabis legalization.

BACKGROUND: As more states legalize cannabis, studies are needed to understand the potential impacts of recreational cannabis legalization (RCL) on adolescents from the perspective of clinicians who care for them. METHODS: This qualitative study characterized clinician perspectives on whether cannabis legalization is associated with changes in adolescents' cannabis use beliefs, behaviors, and consequences. Semi-structured qualitative interviews were conducted with 32 clinicians in a large healthcare organization from 9/6/2022-12/21/2022. Video-recorded interviews were transcribed and analyzed using thematic analysis. RESULTS: The 32 participants (56.3 % female, mean [SD] age, 45.9 [7.6] years; 65.3 % non-Hispanic White) were from Addiction Medicine (n = 13), Psychiatry/Mental Health (n = 7), Pediatrics (n = 5), and the Emergency Department (n = 7). Clinicians described post-RCL increases in adolescent cannabis use, use of non-combustible modes and high-potency products, and younger age of first use. Clinicians reported social, physical, and policy changes, including changes in social norms, appealing advertisements, marketing, and easier access. Many noted fewer perceived harms among adolescents and greater self-medication post-RCL. They described how RCL contributed to increased parental cannabis use and permissiveness around adolescent use. Finally, many described post-RCL increases in cannabis hyperemesis syndrome, and several noted increased cannabis-related psychosis and acute intoxication, and decreased court-mandated treatment. CONCLUSIONS: Clinicians from diverse specialties described post-RCL increases in adolescent cannabis use and cannabis-related consequences, alongside changes in social norms, access, marketing and advertisements, and decreased perceptions of harms. Findings can inform strategies to support adolescents in the context of increased cannabis availability and acceptability post-legalization and support the development of hypotheses for broader-scale quantitative work.

Let's talk about sex: Why reproductive systems matter for understanding algae.

Sex is a crucial process that has molecular, genetic, cellular, organismal, and population-level consequences for eukaryotic evolution. Eukaryotic life cycles are composed of alternating haploid and diploid phases but are constrained by the need to accommodate the phenotypes of these different phases. Critical gaps in our understanding of evolutionary drivers of the diversity in algae life cycles include how selection acts to stabilize and change features of the life cycle. Moreover, most eukaryotes are partially clonal, engaging in both sexual and asexual reproduction. Yet, our understanding of the variation in their reproductive systems is largely based on sexual reproduction in animals or angiosperms. The relative balance of sexual versus asexual reproduction not only controls but also is in turn controlled by standing genetic variability, thereby shaping evolutionary trajectories. Thus, we must quantitatively assess the consequences of the variation in life cycles on reproductive systems. Algae are a polyphyletic group spread across many of the major eukaryotic lineages, providing powerful models by which to resolve this knowledge gap. There is, however, an alarming lack of data about the population genetics of most algae and, therefore, the relative frequency of sexual versus asexual processes. For many algae, the occurrence of sexual reproduction is unknown, observations have been lost in overlooked papers, or data on population genetics do not yet exist. This greatly restricts our ability to forecast the consequences of climate change on algal populations inhabiting terrestrial, aquatic, and marine ecosystems. This perspective summarizes our extant knowledge and provides some future directions to pursue broadly across micro- and macroalgal species.

Hypothesized life cycle of the snow algae Chlainomonas sp. (Chlamydomonadales, Chlorophyta) from the Cascade Mountains, USA.

Chlainomonas (Chlamydomonadales, Chlorophyta) is one of the four genera of snow algae known to produce annual pink or red blooms in alpine snow. No Chlainomonas species have been successfully cultured in the laboratory, but diverse cell types have been observed from many field-collected samples, from multiple species. The diversity of morphologies suggests these algae have complex life cycles with changes in ploidy. Over 7 years (2017-2023), we observed seasonal blooms dominated by a Chlainomonas species from late spring through the summer months on a snow-on-lake habitat in an alpine basin in the North Cascade Mountains of Washington, USA. The Bagley Lake Chlainomonas is distinct from previously reported species based on morphology and sequence data. We observed a similar collection of cell types observed in other Chlainomonas species, with the addition of swarming biflagellate cells that emerged from sporangia. We present a life cycle hypothesis for this species that links cell morphologies observed in the field to seasonally available habitat. The progression of cell types suggests cells are undergoing both meiosis and fertilization in the life cycle. Since the life cycle is the most fundamental biological feature of an organism, with direct consequences for evolutionary processes, it is critical to understand how snow algal life cycles will influence their responses to changes in their habitat driven by climate warming. For microbial taxa that live in extreme environments and are difficult to culture, temporal field studies, such as we report here, may be key to creating testable hypotheses for life cycles.