Retrospective Review of Multitarget Stool DNA as a Screening Test for Colorectal Cancer.

OBJECTIVES: To review the effectiveness of noninvasive multitarget stool DNA testing as a screening test for colorectal cancer. METHODS: We performed a retrospective review of patients referred to 2 high volume outpatient procedural centers for colonoscopy for positive Cologuard test. Positive findings for colorectal cancer based on pathologic findings and also advanced adenomas were recorded. Positive predictive value (PPV) was assessed. RESULTS: Of the 1585 patients evaluated and referred for colonoscopy from January 1, 2018 to November 1, 2019, for ICD-10 codes R19.5 (other fecal abnormalities) and K92.1 (melena), 84 were referred for a positive Cologuard test. Out of the 84, 6 were excluded based on family history of colon cancer in first degree relative or personal history of inflammatory bowel disease. Of the remaining 78 patients, 1 patient (1.3%) had colorectal cancer and 5 (6.4%) had advanced adenomas (>1 cm size, high grade dysplasia or villous). Postive predictive value for colorectal cancer was 1.3% and for precancerous lesions plus colorectal cancer was 7.7%. A total of 53 (68.0%) patients had either totally normal colonoscopy or hyperplastic polyps. Out of the 78 individuals in our study, 70 (89.7%) had normal findings, hyperplastic polyps, or non-advanced adenomas. CONCLUSIONS: Multitarget stool DNA testing carries an unacceptably low PPV to be utilized as a screening test for colorectal cancer. The study fails to detect both adenomas and colon cancer at a higher rate than screening colonoscopy in selected studies. The advantage of being noninvasive has been noted to increase colorectal cancer screening in otherwise non-compliant Medicare patients.

Identifying three otopathologies in humans.

OBJECTIVES: Hearing-in-noise (HIN) is a primary complaint of both the hearing impaired and the hearing aid user. Both auditory nerve (AN) function and outer hair cell (OHC) function are thought to contribute to HIN, but their relative contributions are still being elucidated. OHCs play a critical role in HIN by fine tuning the motion of the basilar membrane. Further, animal studies suggest that cochlear (auditory) synaptopathy, which is the loss of synaptic contact between hair cells and the AN, may be another cause of HIN difficulty. While there is evidence that cochlear synaptopathy occurs in animal models, there is debate as to whether cochlear synaptopathy is clinically significant in humans, which may be due to disparate methods of measuring noise exposure in humans and our high variability in susceptibility to noise damage. Rather than use self-reported noise exposure to define synaptopathic groups, this paper assumes that the general population exhibits a range of noise exposures and resulting otopathologies and defines cochlear synaptopathy "operationally" as low CAP amplitude accompanied by normal DPOAE levels in persons with low pure tone averages. The first question is whether the standard audiogram detects AN dysfunction and OHC dysfunction? The second question is whether HIN performance is primarily dependent on AN function, OHC function, or both functions? DESIGN: Adult subjects have been recruited to participate in an ongoing study and variables such as age, self-reported gender, pure tone audiometry (0.25-20 kHz), subjective perception of HIN difficulty, Quick Speech-in Noise (QuickSIN) test, 45% time compressed word recognition (WR) in 10% reverberation and WR in the presence of ipsilateral speech-weighted noise have been collected. These variables were correlated with OHC function measured by distortion-product otoacoustic emission (DPOAE) signal to-noise-ratio (SNR), and AN function measured by compound action potential (CAP) peak amplitude and ratio to summating potential measured using electrocochleography. RESULTS: Synaptopathy, by this operational definition, may be present in as many as 30% of individuals with normal hearing. Persons hearing within normal limits may exhibit HIN difficulties, and persons with hearing within normal limits may exhibit two distinct types of otopathologies undetected by the standard audiogram (a.k.a. hidden hearing loss) namely operational cochlear synaptopathy and OHC dysfunction. AN untuning secondary to OHC dysfunction is a third otopathology that occurs in subjects with a Mild-Moderate sensorineural hearing loss (SNHL). Clinical norms for each of these otopathologies are presented. Finally, the data show that operational cochlear synaptopathy does not correlate with HIN dysfunction. Rather, HIN performance is primarily governed by OHC function, while AN untuning also plays a lesser but statistically significant role. CONCLUSIONS: The results of this study suggest the following: (1) persons hearing within normal limits may exhibit HIN difficulties; (2) persons hearing within normal limits may exhibit undetected otopathologies, namely AN dysfunction and OHC dysfunction; (3) AN untuning secondary to OHC dysfunction occurs in subjects with Mild-Moderate SNHL; (4) HIN performance is primarily governed by OHC function rather than AN function.

Outer Hair Cell and Auditory Nerve Function in Speech Recognition in Quiet and in Background Noise.

The goal of this study was to describe the contribution of outer hair cells (OHCs) and the auditory nerve (AN) to speech understanding in quiet and in the presence of background noise. Fifty-three human subjects with hearing ranging from normal to moderate sensorineural hearing loss were assayed for both speech in quiet (Word Recognition) and speech in noise (QuickSIN test) performance. Their scores were correlated with OHC function as assessed via distortion product otoacoustic emissions, and AN function as measured by amplitude, latency, and threshold of the VIIIth cranial nerve Compound Action Potential (CAP) recorded during electrocochleography (ECochG). Speech and ECochG stimuli were presented at equivalent sensation levels in order to control for the degree of hearing sensitivity across patients. The results indicated that (1) OHC dysfunction was evident in the lower range of normal audiometric thresholds, which demonstrates that OHC damage can produce "Hidden Hearing Loss," (2) AN dysfunction was evident beginning at mild levels of hearing loss, (3) when controlled for normal OHC function, persons exhibiting either high or low ECochG amplitudes exhibited no statistically significant differences in neither speech in quiet nor speech in noise performance, (4) speech in noise performance was correlated with OHC function, (5) hearing impaired subjects with OHC dysfunction exhibited better speech in quiet performance at or near threshold when stimuli were presented at equivalent sensation levels. These results show that OHC dysfunction contributes to hidden hearing loss, OHC function is required for optimum speech in noise performance, and those persons with sensorineural hearing loss exhibit better word discrimination in quiet at or near their audiometric thresholds than normal listeners.

An independent construct for conditional expression of atonal homolog-1.

The mammalian homolog of the basic helix-loop-helix transcription factor atonal-1 (Atoh1 or Math1) is required for development of cochlear hair cells that function as the mechanosensory cells required for audition. Forced expression of Atoh1 in cochlear-supporting cells may provide a way to regenerate hair cells and provide for a therapy for hearing loss. Additionally, Atoh1 is an inhibitor of proliferation and has further clinical applications in anticancer therapies. The goal of these experiments was to improve the method for Atoh1 expression by engineering a genetic construct that may be used in future translational applications. To address the poor control of Atoh1 expression in standard gene expression systems where Atoh1 is expressed constitutively at abnormally elevated levels, our aim was to engineer an inducible system whereby Atoh1 was upregulated by an inducer and downregulated once the inducer was removed. A further aim was to engineer a single genetic construct that allowed for conditional expression of Atoh1 independent of secondary regulatory elements. Here we describe a stand-alone genetic construct that utilizes the tamoxifen sensitivity of a mutated estrogen receptor (ER) ligand-binding domain for the conditional expression of Atoh1. The Atoh1-ER-DsRed construct is translated into an ATOH1-ER-DSRED fusion protein that remains sequestered in the cytoplasm and therefore rendered inactive because it cannot enter the nucleus to activate Atoh1 signaling pathways. However, application of 4-hydroxytamoxifen results in translocation of the fusion protein to the nucleus, where it binds to the Atoh1 enhancer, upregulates transcription and translation of endogenous ATOH1 and activates downstream Atoh1 signaling such as upregulation of the hair cell protein MYOSIN 7A. Removal of tamoxifen reverses the upregulation of endogenous Atoh1 signaling. This construct serves as an independent genetic construct that allows for the conditional upregulation and downregulation of Atoh1, and may prove useful for manipulating Atoh1 expression in vivo.

TAK1 expression in the cochlea: a specific marker for adult supporting cells.

Transforming growth factor-ß-activated kinase-1 (TAK1) is a mitogen activated protein kinase kinase kinase that is involved in diverse biological roles across species. Functioning downstream of TGF-ß and BMP signaling, TAK1 mediates the activation of the c-Jun N-terminal kinase signaling pathway, serves as the target of pro-inflammatory cytokines, such as TNF-α, mediates NF-κß activation, and plays a role in Wnt/Fz signaling in mesenchymal stem cells. Expression of TAK1 in the cochlea has not been defined. Data mining of previously published murine cochlear gene expression databases indicated that TAK1, along with TAK1 interacting proteins 1 (TAB1), and 2 (TAB2), is expressed in the developing and adult cochlea. The expression of TAK1 in the developing cochlea was confirmed using RT-PCR and immunohistochemistry. Immunolabeling of TAK1 in embryonic, neonatal, and mature cochleas via DAB chromogenic and fluorescent immunohistochemistry indicated that TAK1 is broadly expressed in both the developing otocyst and periotic mesenchyme at E12.5 but becomes more restricted to specific types of supporting cells as the organ of Corti matures. By P1, TAK1 immunolabeling is found in cells of the stria vascularis, hair cells, supporting cells, and Kölliker's organ. By P16, TAK1 labeling is limited to cochlear supporting cells. In the adult cochlea, TAK1 immunostaining is only present in the cytoplasm of Deiters' cells, pillar cells, inner phalangeal cells, and inner border cells, with no expression in any other cochlear cell types. While the role of TAK1 in the inner ear is unclear, TAK1 expression may be used as a novel marker for specific sub-populations of supporting cells.

Biotechnology in the treatment of sensorineural hearing loss: foundations and future of hair cell regeneration.

PURPOSE: To provide an overview of the methodologies involved in the field of hair cell regeneration. First, the author provides a tutorial on the biotechnological foundations of this field to assist the reader in the comprehension and interpretation of the research involved in hair cell regeneration. Next, the author presents a review of stem cell and gene therapy and provides a critical appraisal of their application to hair cell regeneration. The methodologies used in these approaches are highlighted. METHOD: The author conducted a narrative review of the fields of cellular, molecular, and developmental biology, tissue engineering, and stem cell and gene therapy using the PubMed database. RESULTS: The use of biotechnological approaches to the treatment of hearing loss--approaches such as stem cell and gene therapy-has led to new methods of regenerating cochlear hair cells in mammals. CONCLUSIONS: Incredible strides have been made in assembling important pieces of the puzzle that comprise hair cell regeneration. However, mammalian hair cell regeneration using stem cell and gene therapy are years--if not decades--away from being clinically feasible. If the goals of the biological approaches are met, these therapies may represent future treatments for hearing loss.

Neural stem cells injected into the sound-damaged cochlea migrate throughout the cochlea and express markers of hair cells, supporting cells, and spiral ganglion cells.

Most cases of hearing loss are caused by the death or dysfunction of one of the many cochlear cell types. We examined whether cells from a neural stem cell line could replace cochlear cell types lost after exposure to intense noise. For this purpose, we transplanted a clonal stem cell line into the scala tympani of sound damaged mice and guinea pigs. Utilizing morphological, protein expression and genetic criteria, stem cells were found with characteristics of both neural tissues (satellite, spiral ganglion, and Schwann cells) and cells of the organ of Corti (hair cells, supporting cells). Additionally, noise-exposed, stem cell-injected animals exhibited a small but significant increase in the number of satellite cells and Type I spiral ganglion neurons compared to non-injected noise-exposed animals. These results indicate that cells of this neural stem cell line migrate from the scala tympani to Rosenthal's canal and the organ of Corti. Moreover, they suggest that cells of this neural stem cell line may derive some information needed from the microenvironment of the cochlea to differentiate into replacement cells in the cochlea.

Regeneration and replacement in the vertebrate inner ear.

Deafness affects more than 40 million people in the UK and the USA, and many more world-wide. The primary cause of hearing loss is damage to or death of the sensory receptor cells in the inner ear, the hair cells. Birds can readily regenerate their cochlear hair cells but the mammalian cochlea has shown no ability to regenerate after damage. Current research efforts are focusing on gene manipulation, gene therapy and stem cell transplantation for repairing or replacing damaged mammalian cochlear hair cells, which could lead to therapies for treating deafness in humans.

Expression profile of an operationally-defined neural stem cell clone.

Neural stem cells (NSCs) are the most primordial and least committed cells of the nervous system, the cells that exist before regional specification develops. Because immunocytochemically-detectable markers that are sufficiently specific and sensitive to define an NSC have not yet been fully defined, we have taken the strong view that, to be termed a "stem cell" in the nervous system--in contrast to a "progenitor" or "precursor" (whose lineage commitment is further restricted)--a single neuroectodermally-derived cell must fulfill an operational definition that is essentially similar to that used in hematopoiesis. In other words, it must possess the following functional properties: (1) "Multipotency", i.e., the ability to yield mature cells in all three fundamental neural lineages throughout the nervous system--neurons (of all subtypes), astrocytes (of all types), oligodendrocytes--in multiple regional and developmental contexts and in a region and developmental stage-appropriate manner. (2) The ability to populate a developing region and/or repopulate an ablated or degenerated region of the nervous system with appropriate cell types. (3) The ability to be serially transplanted. (4) "Self-renewal", i.e., the ability to produce daughter cells (including new NSCs) with identical properties and potential. Having identified a murine neural cell clone that fulfills this strict operational definition--in contrast to other studies that used less rigorous or non-operational criteria for defining an NSC (e.g., the "neurosphere" assay)--we then examined, by comparing gene expression profiles, the relationship such a cell might have to (a) a multipotent somatic stem cell from another organ system (the hematopoietic stem cell [HSC]); (b) a pluripotent stem cell derived from the inner cell mass and hence without organ assignment (an embryonic stem cell); (c) neural cells isolated and maintained primarily as neurospheres but without having been subjected to the above mentioned operational screen ("CNS-derived neurospheres"). ESCs, HSCs, and operationally-defined NSCs--all of which have been identified not only by markers but by functional assays in their respective systems and whose state of differentiation could be synchronized--shared a large number of genes. Although, as expected, the most stem-like genes were expressed by ESCs, NSCs and HSCs shared a number of genes. CNS-derived neurospheres, on the other hand, expressed fewer "stem-like" genes held in common by the other operationally-defined stem cell populations. Rather they displayed a profile more consistent with differentiated neural cells. (Genes of neural identity were shared with the NSC clone.) Interestingly, when the operationally-defined NSC clone was cultured as a neurosphere (rather than in monolayer), its expression pattern shifted from a "stem-like" pattern towards a more "differentiated" one, suggesting that the neurosphere, without functional validation, may be a poor model for predicting stem cell attributes because it consists of heterogeneous populations of cells, only a small proportion of which are truly "stem-like". Furthermore, when operational definitions are employed, a common set of stem-like genes does emerge across both embryonic and somatic stem cells of various organ systems, including the nervous system.

Cell contact-dependent mechanisms specify taste bud pattern during a critical period early in embryonic development.

After gastrulation, the pharyngeal endoderm is specified to give rise to taste receptor organs without further signaling from other embryonic tissues. We hypothesized that intercellular signaling might be responsible for the specification of taste buds. To test if and when this signaling was occurring, intercellular contacts were transiently disrupted in cultures of pharyngeal endoderm from axolotl embryos, and the number, size, and distribution of taste buds analyzed. Disruption of cell contacts at progressive time points, from neurula to late tail bud stages, revealed a critical period, during mid-tail bud stages, when disruption of cell contacts resulted in a significant increase in taste bud number and size. The spatial distribution of taste buds was also altered; taste buds were more clustered in explants disrupted during the critical period. These effects were not due to general alterations in mitosis and apoptosis. Rather, at least three aspects of taste bud patterning, i.e., number, size, and distribution, are governed by mechanisms dependent on normal cell contacts during a concise time window. Furthermore, our findings are consistent with specification of taste buds by means of lateral inhibitory signaling, which we hypothesize results from cell contact-dependent or short-range diffusible signals.

The potential use of stem cells for cochlear repair.

In light of the currently defined characteristics of stem cells, a re-evaluation of hair cell regeneration in birds suggests that there may be a stem cell population located in the inner ear. It is yet to be determined if the mammalian cochlea contains stem cells, but the presence of a mammalian vestibular stem cell population would not appear to be out of the realm of possibility. This paper reviews the latest advances in stem cell biology and suggests that stem cells may be an appropriate biological tool to be used for cochlear repair. The potential use of several types of stem cells, including embryonic, neural and hematopoietic stem cells, as agents for cochlear repair is examined.

Platelet-activating factor induces permeability transition and cytochrome c release in isolated brain mitochondria.

Platelet-activating factor (PAF), a potent bioactive phospholipid implicated in neuronal excitotoxic death, was assessed as a mediator of brain mitochondrial dysfunction. Carbamyl PAF, a non-hydrolyzable PAF analog, added to neurons in culture resulted in decreased mitochondrial membrane potential (DeltaPsi(M)) as measured by the DeltaPsi(M)-sensitive fluorophore 5,5', 6,6'-tetrachloro-1, 1', 3,3'-tetraethylethylbenzimidazolo-carbocyanide iodide (JC-1). To investigate whether PAF has a direct effect on the mitochondria, the mediator was added to rat brain mitochondria preparations and an increase in the permeability of the mitochondrial membrane, termed permeability transition (PT), and cytochrome c release were measured. We report that PAF causes both dose-dependent PT and cytochrome c release from isolated mitochondria. Furthermore, the selective PAF antagonist tetrahydro-4,7,8,10 methyl-1 (chloro-2 phenyl)-6 (methoxy-4 phenyl-carbamoyl)-9 pyrido [4',3'-4,5] thieno [3,2-f] triazolo-1,2,4 [4,3-a] diazepine-1,4 (BN50730), which has affinity for intracellular binding sites, and the peripheral benzodiazepine receptor ligands 7-chloro-5- [4'-chlorophenyl]-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864) and 1-(-2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), inhibit PAF induction of PT and cytochrome c release. These results suggest that PAF excitotoxicity involves, at least in part, alterations of the mitochondrial membrane.