Purification and characterization of human neural stem and progenitor cells.

The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.

Constant Sub-second Cycling between Representations of Possible Futures in the Hippocampus.

Cognitive faculties such as imagination, planning, and decision-making entail the ability to represent hypothetical experience. Crucially, animal behavior in natural settings implies that the brain can represent hypothetical future experience not only quickly but also constantly over time, as external events continually unfold. To determine how this is possible, we recorded neural activity in the hippocampus of rats navigating a maze with multiple spatial paths. We found neural activity encoding two possible future scenarios (two upcoming maze paths) in constant alternation at 8 Hz: one scenario per ∼125-ms cycle. Further, we found that the underlying dynamics of cycling (both inter- and intra-cycle dynamics) generalized across qualitatively different representational correlates (location and direction). Notably, cycling occurred across moving behaviors, including during running. These findings identify a general dynamic process capable of quickly and continually representing hypothetical experience, including that of multiple possible futures.

Ets2 anchors the prometastatic function of mutant p53 in osteosarcoma.

Mutations in the tumor suppressor p53 occur in a majority of human cancers. Some gain-of-function (GOF) p53 mutations endow tumor cells with increased metastatic ability, although our understanding of the underlying mechanism remains incomplete. In this issue of Genes & Development, Pourebrahim and colleagues (pp. 1847-1857) develop a new mouse model of osteosarcoma in which a GOF mutant p53 allele is expressed specifically in osteoblasts, while the tumor microenvironment remains wild type for p53, allowing for the study of cell-autonomous functions. In this model, the role of GOF mutant p53 in promoting lung metastasis is shown to be critically dependent on the transcription factor Ets2 and is accompanied by the elevated expression of a cluster of small nucleolar RNAs (snoRNAs).

Identification of Nidogen 1 as a lung metastasis protein through secretome analysis.

Secreted proteins play crucial roles in mediating tumor-stroma interactions during metastasis of cancer to different target organs. To comprehensively profile secreted proteins involved in lung metastasis, we applied quantitative mass spectrometry-based proteomics and identified 392 breast cancer-derived and 302 melanoma-derived proteins secreted from highly lung metastatic cells. The cancer-specific lung metastasis secretome signatures (LMSSs) displayed significant prognostic value in multiple cancer clinical data sets. Moreover, we observed a significant overlap of enriched pathways between the LMSSs of breast cancer and melanoma despite an overall small overlap of specific proteins, suggesting that common biological processes are executed by different proteins to enable the two cancer types to metastasize to the lung. Among the novel candidate lung metastasis proteins, Nidogen 1 (NID1) was confirmed to promote lung metastasis of breast cancer and melanoma, and its expression is correlated with poor clinical outcomes. In vitro functional analysis further revealed multiple prometastatic functions of NID1, including enhancing cancer cell migration and invasion, promoting adhesion to the endothelium and disrupting its integrity, and improving vascular tube formation capacity. As a secreted prometastatic protein, NID1 may be developed as a new biomarker for disease progression and therapeutic target in breast cancer and melanoma.

Block Aligner: an adaptive SIMD-accelerated aligner for sequences and position-specific scoring matrices.

MOTIVATION: Efficiently aligning sequences is a fundamental problem in bioinformatics. Many recent algorithms for computing alignments through Smith-Waterman-Gotoh dynamic programming (DP) exploit Single Instruction Multiple Data (SIMD) operations on modern CPUs for speed. However, these advances have largely ignored difficulties associated with efficiently handling complex scoring matrices or large gaps (insertions or deletions). RESULTS: We propose a new SIMD-accelerated algorithm called Block Aligner for aligning nucleotide and protein sequences against other sequences or position-specific scoring matrices. We introduce a new paradigm that uses blocks in the DP matrix that greedily shift, grow, and shrink. This approach allows regions of the DP matrix to be adaptively computed. Our algorithm reaches over 5-10 times faster than some previous methods while incurring an error rate of less than 3% on protein and long read datasets, despite large gaps and low sequence identities. AVAILABILITY AND IMPLEMENTATION: Our algorithm is implemented for global, local, and X-drop alignments. It is available as a Rust library (with C bindings) at https://github.com/Daniel-Liu-c0deb0t/block-aligner.

MRI-based vertebral bone quality score: relationship with age and reproducibility.

Vertebral bone quality (VBQ) score is an opportunistic measure of bone mineral density using routine preoperative MRI in spine surgery. VBQ score positively correlates with age and is reproducible across serial scans. However, extrinsic factors, including MRI machine and protocol, affect the VBQ score and must be standardized. PURPOSE: The purposes of this study were to determine whether VBQ score increased with age and whether VBQ remained consistent across serial MRI studies obtained within 3 months. METHODS: This retrospective study evaluated 136 patients, age 20-69, who received two T1-weighted lumbar MRI within 3 months of each other between January 2011 and December 2021. VBQ(L1-4) score was calculated as the quotient of L1-L4 signal intensity (SI) and L3 cerebral spinal fluid (CSF) SI. VBQ(L1) score was calculated as the quotient of L1 SI and L1 CSF SI. Regression analysis was performed to determine correlation of VBQ(L1-4) score with age. Coefficient of variation (CV) was used to determine reproducibility between VBQ(L1-4) scores from serial MRI scans. RESULTS: One hundred thirty-six patients (mean ± SD age 44.9 ± 12.5 years; 53.7% female) were included in this study. Extrinsic factors affecting the VBQ score included patient age, MRI relaxation time, and specific MRI machine. When controlling for MRI relaxation/echo time, the VBQ(L1-4) score was positively correlated with age and had excellent reproducibility in serial MRI with CV of 0.169. There was excellent agreement (ICC > 0.9) of VBQ scores derived from the two formulas, VBQ(L1) and VBQ(L1-4). CONCLUSION: Extrinsic factors, including MRI technical factors and age, can impact the VBQ(L1-4) score and must be considered when using this tool to estimate bone mineral density (BMD). VBQ(L1-4) score was positively correlated with age. Reproducibility of the VBQ(L1-4) score across serial MRI is excellent especially when controlling for technical factors, supporting use of the VBQ score in estimating BMD. The VBQ(L1) score was a reliable alternative to the VBQ(L1-4) score.

Subtotal Fenestrating Cholecystectomy: A Safe and Effective Approach to the Difficult Gallbladder.

INTRODUCTION: Subtotal laparoscopic cholecystectomy (SUB) is an alternative to total laparoscopic cholecystectomy (TOT) when the critical view of safety (CVS) cannot be achieved. Little is known about the clinical factors and postoperative outcomes associated with SUB. The objective was to determine predictive factors and outcomes of SUB as compared to TOT. METHODS: Clinical data from patients admitted from our emergency department to the acute care surgery service who underwent SUB or TOT by an acute care surgery surgeon for acute biliary disease (2017-2019) were reviewed. Wilcoxon rank-sum and Fisher's exact tests were used. RESULTS: 355 patients underwent cholecystectomy for acute cholecystitis; 28 were SUB (7.9%). SUB patients were more likely to be older (57 versus 43 y; P = 0.015), male (60.7% versus 39.3%; P < 0.001), have a history of cirrhosis or liver disease (14.3% versus 2.1%; P = 0.007), and have a higher Charlson-Comorbidity Index (1 versus 0, P = 0.041). SUB had greater leukocytosis (14.6 versus 10.9; P < 0.001), higher total bilirubin (0.9 versus 0.6; P = 0.021), and a higher Tokyo grade (2 versus 1; P < 0.001), and had operative findings including gallbladder decompression (82.1% versus 23.2%; P < 0.001) and inability to achieve the CVS (78.6% versus 3.4%; P < 0.001). SUB patients had an increased length of stay (4 versus 2 d; P < 0.001) and more 1-y readmissions. No major vascular injuries occurred in either group with one biliary injury in the TOT group. CONCLUSIONS: SUB patients present with more significant markers of biliary disease and have more complicated intraoperative and postoperative courses. However, the lack of biliary or vascular injuries suggests that SUB may represent a safe alternative when the CVS cannot be achieved.

Establishing a pediatric interventional radiology inpatient consult service.

OBJECTIVE: To delineate pediatric interventional radiology (IR) inpatient consult growth and resulting collections after implementation of a pediatric IR consult service. METHODS: An inpatient IR consult process was created at a single academic children's hospital in October 2019. IR consult note templates were created in Epic (Epic Systems Corporation, Verona, Wisconsin) and utilized by 4 IR physicians. Automatic charge generation was linked to differing levels of evaluation and management (E&M) service relating to current procedural terminology (CPT) inpatient consult codes 99251-99255. The children's hospital informatics division identified IR consult notes entered from the implementation of the consult service: October 2019 to January 2022. The university radiology department billing office provided IR service E&M charge, payment, and relative value units (RVU) information during this study period. A chart review was performed to determine the IR procedure conversion rate. Mann-Whitney and a two-sample t-test statistical analyses compared use of the 25-modifier, monthly consult growth and monthly payment growth. P-value < 0.05 was considered statistically significant.  RESULTS: Within this 27-month period, a total of 2153 inpatient IR consults were performed during 1757 Epic hospital encounters; monthly consult peak was reached 5 months into the study period. Consult level breakdown by CPT codes: 99251-8.7%, 99252-81.7%, and 99253-8.8%. 69.7% of IR consults had consult-specific billing with payments in 96.4% resulting in $143,976 new revenue. From 2020 to 2021, IR consult volume trended upward by 13.4% (P =0.069), and consult-specific payments increased by 84.1% (P<0.001). IR consult procedure conversion rate was 96.5%. CONCLUSION: An inpatient pediatric IR consult service was quickly established and maintained by four physicians over a 27-month study period. Annual IR consult volume trended upward and consult-specific payments increased, resulting in previously uncaptured IR service revenue.

Bile Microbiome Signatures Associated with Pancreatic Ductal Adenocarcinoma Compared to Benign Disease: A UK Pilot Study.

Pancreatic ductal adenocarcinoma (PDAC) has a very poor survival. The intra-tumoural microbiome can influence pancreatic tumourigenesis and chemoresistance and, therefore, patient survival. The role played by bile microbiota in PDAC is unknown. We aimed to define bile microbiome signatures that can effectively distinguish malignant from benign tumours in patients presenting with obstructive jaundice caused by benign and malignant pancreaticobiliary disease. Prospective bile samples were obtained from 31 patients who underwent either Endoscopic Retrograde Cholangiopancreatography (ERCP) or Percutaneous Transhepatic Cholangiogram (PTC). Variable regions (V3-V4) of the 16S rRNA genes of microorganisms present in the samples were amplified by Polymerase Chain Reaction (PCR) and sequenced. The cohort consisted of 12 PDAC, 10 choledocholithiasis, seven gallstone pancreatitis and two primary sclerosing cholangitis patients. Using the 16S rRNA method, we identified a total of 135 genera from 29 individuals (12 PDAC and 17 benign). The bile microbial beta diversity significantly differed between patients with PDAC vs. benign disease (Permanova p = 0.0173). The separation of PDAC from benign samples is clearly seen through unsupervised clustering of Aitchison distance. We found three genera to be of significantly lower abundance among PDAC samples vs. benign, adjusting for false discovery rate (FDR). These were Escherichia (FDR = 0.002) and two unclassified genera, one from Proteobacteria (FDR = 0.002) and one from Enterobacteriaceae (FDR = 0.011). In the same samples, the genus Streptococcus (FDR = 0.033) was found to be of increased abundance in the PDAC group. We show that patients with obstructive jaundice caused by PDAC have an altered microbiome composition in the bile compared to those with benign disease. These bile-based microbes could be developed into potential diagnostic and prognostic biomarkers for PDAC and warrant further investigation.

Characteristics, outcomes, and 30-day readmissions following pediatric extracorporeal membrane oxygenation in the United States: A Nationwide Readmissions Database study.

OBJECTIVES: Extracorporeal membrane oxygenation (ECMO) is an increasingly used mode of critical care support for pediatric patients refractory to conventional therapy. We evaluated the characteristics, outcomes, and readmissions rates for pediatric ECMO in the United States. METHODS: Data was extracted from the Nationwide Readmissions Database, a database designed to support national readmissions analyses, for patients aged 1-18 years undergoing ECMO between 2012-2018. Baseline demographics, comorbidities, and characteristics were identified using International Classification of Diseases codes. RESULTS: Out of 897,117 index pediatric hospitalizations, 3706 patients underwent ECMO [median age 9 years (IQR 2,15); 51.6% males]. 2246 (60.6%) patients survived to hospital discharge, with a 30-day readmissions rate of 17% among survivors. Cardiac conditions associated with ECMO were congenital heart disease (25.3%), cardiogenic shock (23.6%), and congestive heart failure (16.2%). The common respiratory associations were sepsis (36.2%), pneumonia (35.6%), and asthma (15.4%). Patients who survived were more likely to have diagnoses of asthma, bronchiolitis, myocarditis, pneumonia, and sepsis. Acute kidney injury (51.5%), disseminated intravascular coagulation (22.5%), and surgical site bleeding (12.7%) were the commonly associated complications. The trend for yearly survival rates was not statistically significant (linear p-trend = 0.38). CONCLUSIONS: Pediatric ECMO continues to be associated with notable mortality and complication rates. We did not observe a meaningful trend for the yearly survival rates over the study period, and over one-sixth of survivors were readmitted within 30-days. More research is needed to identify patients at high risk of mortality and readmissions, to help target resources more efficiently and improve survival.

The Clinical Significance of Transfer RNAs Present in Extracellular Vesicles.

Extracellular vesicles (EVs) are important for intercellular signalling in multi-cellular organisms. However, the role of mature transfer RNAs (tRNAs) and tRNA fragments in EVs has yet to be characterised. This systematic review aimed to identify up-to-date literature on tRNAs present within human EVs and explores their potential clinical significance in health and disease. A comprehensive and systematic literature search was performed, and the study was conducted in accordance with PRISMA guidelines. Electronic databases MEDLINE and EMBASE were searched up until 1 January 2022. From 685 papers, 60 studies were identified for analysis. The majority of papers reviewed focussed on the role of EV tRNAs in cancers (31.7%), with numerous other conditions represented. Blood and cell lines were the most common EV sources, representing 85.9% of protocols used. EV isolation methods included most known methods, precipitation being the most common (49.3%). The proportion of EV tRNAs was highly variable, ranging between 0.04% to >95% depending on tissue source. EV tRNAs are present in a multitude of sources and show promise as disease markers in breast cancer, gastrointestinal cancers, and other diseases. EV tRNA research is an emerging field, with increasing numbers of papers highlighting novel methodologies for tRNA and tRNA fragment discovery.

The molecular vista: current perspectives on molecules and life in the twentieth century.

This essay considers how scholarly approaches to the development of molecular biology have too often narrowed the historical aperture to genes, overlooking the ways in which other objects and processes contributed to the molecularization of life. From structural and dynamic studies of biomolecules to cellular membranes and organelles to metabolism and nutrition, new work by historians, philosophers, and STS scholars of the life sciences has revitalized older issues, such as the relationship of life to matter, or of physicochemical inquiries to biology. This scholarship points to a novel molecular vista that opens up a pluralist view of molecularizations in the twentieth century and considers their relevance to current science.

Dynamic Changes in Soil Microbial Communities with Glucose Enrichment in Sediment Microbial Fuel Cells.

To investigate soil microbial community dynamics in sediment microbial fuel cells (MFCs), this study applied nonhydric (D) and hydric (S) soils to single-chamber and mediator-free MFCs. Glucose was also used to enrich microorganisms in the soils. The voltage outputs of both the D and S sediment MFCs increased over time but differed from each other. The initial open circuit potentials were 345 and 264 mV for the D and S MFCs. The voltage output reached a maximum of 503 and 604 mV for D and S on days 125 and 131, respectively. The maximum power densities of the D and S MFCs were 2.74 and 2.12 mW m-2, analyzed on day 50. Clustering results revealed that the two groups did not cluster after glucose supplementation and 126 days of MFC function. The change in Geobacter abundance was consistent with the voltage output, indicating that these bacteria may act as the main exoelectrogens on the anode. Spearman correlation analysis demonstrated that, in the D soils, Geobacter was positively correlated with Dialister and negatively correlated with Bradyrhizobium, Kaistobacter, Pedomicrobium, and Phascolarctobacterium; in the S soils, Geobacter was positively correlated with Shewanella and negatively correlated with Blautia. The results suggested that different soil sources in the MFCs and the addition of glucose as a nutrient produced diverse microbial communities with varying voltage output efficiencies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00959-x.

Crizotinib sensitizes the erlotinib resistant HCC827GR5 cell line by influencing lysosomal function.

In non-small cell lung cancer, sensitizing mutations in epidermal growth factor receptor (EGFR) or cMET amplification serve as good biomarkers for targeted therapies against EGFR or cMET, respectively. Here we aimed to determine how this different genetic background would affect the interaction between the EGFR-inhibitor erlotinib and the cMET-inhibitor crizotinib. To unravel the mechanism of synergy we investigated the effect of the drugs on various parameters, including cell cycle arrest, migration, protein phosphorylation, kinase activity, the expression of drug efflux pumps, intracellular drug concentrations, and live-cell microscopy. We observed additive effects in EBC-1, H1975, and HCC827, and a strong synergism in the HCC827GR5 cell line. This cell line is a clone of the HCC827 cells that harbor an EGFR exon 19 deletion and has been made resistant to the EGFR-inhibitor gefitinib, resulting in cMET amplification. Remarkably, the intracellular concentration of crizotinib was significantly higher in HCC827GR5 compared to the parental HCC827 cell line. Furthermore, live-cell microscopy with a pH-sensitive probe showed a differential reaction of the pH in the cytoplasm and the lysosomes after drug treatment in the HCC827GR5 in comparison with the HCC827 cells. This change in pH could influence the process of lysosomal sequestration of drugs. These results led us to the conclusion that lysosomal sequestration is involved in the strong synergistic reaction of the HCC827GR5 cell line to crizotinib-erlotinib combination. This finding warrants future clinical studies to evaluate whether genetic background and lysosomal sequestration could guide tailored therapeutic interventions.

HSP70-Mediated NLRP3 Inflammasome Suppression Underlies Reversal of Acute Kidney Injury Following Extracellular Vesicle and Focused Ultrasound Combination Therapy.

Acute kidney injury (AKI) is the abrupt loss of renal function, for which only supportive therapies exist. Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have been shown to be therapeutically effective in treating AKI by spurring endogenous cell proliferation and survival while suppressing inflammation. Pre-treating kidneys with pulsed focused ultrasound (pFUS) has also been shown to enhance MSC therapy for AKI, but its role in MSC-derived EV therapy remains unexplored. Using a mouse model of cisplatin-induced AKI, we show that combination therapy with pFUS and EVs restores physiological and molecular markers of kidney function, more so than either alone. Both pFUS and EVs downregulate heat shock protein 70 (HSP70), the NLRP3 inflammasome, and its downstream pro-inflammatory cytokines IL-1ß and IL-18, all of which are highly upregulated in AKI. In vitro knockdown studies suggest that HSP70 is a positive regulator of the NLRP3 inflammasome. Our study therefore demonstrates the ability of pFUS to enhance EV therapy for AKI and provides further mechanistic understanding of their anti-inflammatory and regenerative effects.

Trends in the characteristics of human functional genomic data on the gene expression omnibus, 2001-2017.

The gene expression omnibus (GEO) is the world's largest public repository of functional genomic data. Despite its broad use in secondary genomic analyses, the temporal trends in the characteristics of genomic data on GEO, including experimental procedures, geographic origin, funder(s), and related disease, have not been examined. We identified 75,376 Series deposited to the GEO during 2001-2017 and built a database of all human genomic data (39,076 Series, 51.8% of all Series). Using the associated publications, we obtained funding information and identified the related disease area. Of the Series with classified disease areas, the two most common were cancer (n = 12,688, 32.5%) and immunologic diseases (n = 2,393, 6.1%), while the percentages of all other disease areas were below 5%, including neurological diseases (n = 1733, 4.4%), infectious diseases (n = 1225, 3.1%), diabetes (n = 828, 2.1%), and cardiovascular diseases (n = 299, 0.8%). In recent years, there has been a significant increase in the use of high-throughput sequencing (HTS), protein array and multiple-platform technologies, as well as in the proportion of North American deposits. Compared to those from other regions, North American deposits appeared to lead the shift from array-based to HTS technologies (odds ratio [OR], 95% confidence intervals [CI] = 3.39, 3.23-3.55, P = 9.40E-323), and were less likely to focus on a major disease area (OR = 0.64, 95% CI: 0.61-0.67, P = 5.02E-107), suggesting a greater emphasis on basic science in North America. Furthermore, the Series utilizing HTS were less likely to be disease-classified compared to other technologies (OR = 0.39, 95% CI: 0.37-0.41, P = 1.00E-322), suggesting a preferential use or adoption of HTS in basic science settings. Finally, funding from the NHGRI, NCI, NIEHS, and NCCR resulted in a higher number of GEO Series per grant than other NIH institutes, demonstrating different preferences on genomic studies among awardees of NIH institutes. Our findings demonstrate geographic, technological, and funding disparities in the trends of GEO deposit characteristics.

Carbon Dioxide-Mediated C(sp3)-H Arylation of Amine Substrates.

Elaborating amines via C-H functionalization has been an important area of research over the past decade but has generally relied on an added directing group or sterically hindered amine approach. Since free-amine-directed C(sp3)-H activation is still primarily limited to cyclization reactions and to improve the sustainability and reaction scope of amine-based C-H activation, we present a strategy using CO2 in the form of dry ice that facilitates intermolecular C-H arylation. This methodology has been used to enable an operationally simple procedure whereby 1° and 2° aliphatic amines can be arylated selectively at their γ-C-H positions. In addition to potentially serving as a directing group, CO2 has also been demonstrated to curtail the oxidation of sensitive amine substrates.

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.

Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

The Cell and Protoplasm as Container, Object, and Substance, 1835-1861.

This article revisits the development of the protoplasm concept as it originally arose from critiques of the cell theory, and examines how the term "protoplasm" transformed from a botanical term of art in the 1840s to the so-called "living substance" and "the physical basis of life" two decades later. I show that there were two major shifts in biological materialism that needed to occur before protoplasm theory could be elevated to have equal status with cell theory in the nineteenth century. First, I argue that biologists had to accept that life could inhere in matter alone, regardless of form. Second, I argue that in the 1840s, ideas of what formless, biological matter was capable of dramatically changed: going from a "coagulation paradigm" (Pickstone, 1973) that had existed since Theophrastus, to a more robust conception of matter that was itself capable of movement and self-maintenance. In addition to revisiting Schleiden and Schwann's original writings on cell theory, this article looks especially closely at Hugo von Mohl's definition of the protoplasm concept in 1846, how it differed from his primordial utricle theory of cell structure two years earlier. This article draws on Lakoff and Johnson's theory of "ontological metaphors" to show that the cell, primordial utricle, and protoplasm can be understood as material container, object, and substance, and that these overlapping distinctions help explain the chaotic and confusing early history of cell theory.