Tests of human auditory temporal resolution: preliminary investigation of ZEST parameters for amplitude modulation detection.

Auditory temporal resolution plays a critical role in the everyday experience of listening to complex acoustic patterns. Amplitude modulation detection thresholds are widely used to measure auditory temporal resolution. In an attempt to develop a standardized clinical test of auditory temporal resolution, we used ZEST (Zippy Estimation by Sequential Testing, a Bayesian threshold estimation procedure, to measure amplitude modulation detection thresholds. ZEST utilizes prior knowledge about a listener's thresholds, as represented by a probability density function of the thresholds, and psychometric functions of the listener's responses. This paper reports a preliminary study in which ZEST parameters that could be used for measurements of amplitude modulation detection thresholds were sought. For this purpose, we created histograms of the detection thresholds for a wide range of modulation frequencies, measured the psychometric functions of amplitude modulation detection, and performed computer simulations of ZEST threshold estimation. The results suggested that, with appropriately-set parameters, ZEST allows for the accurate estimation of amplitude modulation detection thresholds within 20 trials.

Phenotype-genotype correlation in patients with typical and atypical branchio-oto-renal syndrome.

Some patients have an atypical form of branchio-oto-renal (BOR) syndrome, which does not satisfy the diagnostic criteria, despite carrying a pathogenic variant (P variant) or a likely pathogenic variant (LP variant) of a causative gene. P/LP variants phenotypic indices have yet to be determined in patients with typical and atypical BOR syndrome. We hypothesized that determining phenotypic and genetic differences between patients with typical and atypical BOR syndrome could inform such indices. Subjects were selected from among patients who underwent genetic testing to identify the cause of hearing loss. Patients were considered atypical when they had two major BOR diagnostic criteria, or two major criteria and one minor criterion; 22 typical and 16 atypical patients from 35 families were included. Genetic analysis of EYA1, SIX1, and SIX5 was conducted by direct sequencing and multiplex ligation-dependent probe amplification. EYA1 P/LP variants were detected in 25% and 86% of atypical and typical patients, respectively. Four EYA1 P/LP variants were novel. Branchial anomaly, inner ear anomaly, and mixed hearing loss were correlated with P/LP variants. Development of refined diagnostic criteria and phenotypic indices for atypical BOR syndrome will assist in effective detection of patients with P/LP variants among those with suspected BOR syndrome.

Anticipating real-ear insertion response using an external auditory canal model.

OBJECTIVES: This study aimed to determine the acoustic characteristics of the external auditory canal (EAC) and predict the real-ear aided response (REAR) using an EAC model that includes the standing wave effect. METHODS: The EAC transfer function equations were derived by summing the incoming and outgoing waves. First, we investigated the real-ear unaided gain (REUG). Second, seven patients (eight ears) wearing hearing aids (HAs) were enrolled as subjects to examine the REAR. We conducted wideband tympanometry (WBT) to measure the absorbance, the frequency response at 65 dB (65dB-FR) of the HAs, and the measured REAR for an international speech test signal (ISTS) at 65 dB. RESULTS: The EAC model that includes the standing-wave effect is considered to be valid from examination of the REUG. A significant correlation was found between the measured and calculated REARs at 900 Hz, 1000 Hz, 2000 Hz, and 3000 Hz in an uncorrelated test. A two-way analysis of variance (ANOVA) found significant differences in the 65dB-FR and the measured REARs at 800, 900, 1000, and 2000 Hz, but this difference disappeared after correction of the calculated acoustic characteristics of the EAC. CONCLUSIONS: By measuring the WBT characteristics and correcting them with an EAC model, the in-situ REAR can be determined from the HA characteristics in the mid-frequency range. There is a risk of insufficient HA amplification in the mid-frequency range when no real-ear measurements are performed.

[A Case of Advanced Esophageal Cancer Discovered Due to Suffocation, Which Was Invasive to the Trachea and Internal Carotid Artery and Treated by Chemotherapy].

A 51-year-old male presented with dyspnea due to upper airway obstruction. We decided to perform a cricothyroidotomy due to difficulty in performing orotracheal intubation. A CT scan revealed a massive tumor infiltrating into the right side of the neck, which penetrated the internal carotid artery. An upper gastrointestinal tract endoscopy was performed, and the patient was diagnosed with advanced esophageal cancer(stage Ⅳ, cT4N4M0). We initiated palliative chemotherapy of FOLFOX as first-line chemotherapy. After the fourth course, the patient was evaluated as having progressive disease(PD)due to regrowth of lymph node metastasis around the lower esophagus. Although we changed the treatment to nivolumab as second-line chemotherapy, there was a gradual exacerbation of airway obstruction, and the head and upper limb edema emerged due to superior vena cava syndrome. After the first course of nivolumab, we diagnosed the patient as having clinically PD. After the first course of docetaxel(DTX)as third-line chemotherapy, he suddenly died of massive hemorrhage caused by the intubation tube on day 136. Airway management is difficult to perform in patients with a poor response to chemotherapy due to obstruction by a tumor. On the other hand, excessive response to chemotherapy is also associated with a risk of massive hemorrhage due to arterial perforation, as observed in this case.

Rising-frequency chirp stimulus to effectively enhance wave-I amplitude of auditory brainstem response.

This study aims to find an effective chirp signal that enhances the amplitude of wave-I of auditory brainstem response (ABR) to diagnose "cochlear synaptopathy." Although several chirp signals have been proposed to enhance the amplitude of wave-V, the effect on wave-I has not been clarified yet. Ten chirp signals, which have shorter group delays than the commonly used "CE-chirp," were produced to measure the amplitudes of wave-I and wave-V of the ABRs. The results show that one of the chirp signals significantly enhanced the amplitude of wave-I, where the group delay is approximately half of the CE-chirp.

Deterioration in Distortion Product Otoacoustic Emissions in Auditory Neuropathy Patients With Distinct Clinical and Genetic Backgrounds.

OBJECTIVES: Auditory neuropathy (AN) is a clinical disorder characterized by the absence of auditory brainstem response and presence of otoacoustic emissions. A gradual loss of otoacoustic emissions has been reported for some cases of AN. Such cases could be diagnosed as cochlear hearing loss and lead to misunderstanding of the pathology when patients first visit clinics after the loss of otoacoustic emissions. The purpose of this study was to investigate the time course of changes in distortion product otoacoustic emissions (DPOAEs) in association with patients' genetic and clinical backgrounds, including the use of hearing aids. DESIGN: DPOAE measurements from 31 patients with AN were assessed. Genetic analyses for GJB2, OTOF, and mitochondrial m.1555A> G and m.3243A> G mutations were conducted for all cases, and the analyses for CDH23 and OPA1 were conducted for the selected cases. Patients who were younger than 10 years of age at the time of AN diagnosis were designated as the pediatric AN group (22 cases), and those who were 18 years of age or older were designated as the adult AN group (9 cases). DPOAE was measured at least twice in all patients. The response rate for DPOAEs was defined and analyzed. RESULTS: The pediatric AN group comprised 10 patients with OTOF mutations, 1 with GJB2 mutations, 1 with OPA1 mutation, and 10 with indefinite causes. Twelve ears (27%) showed no change in DPOAE, 20 ears (46%) showed a decrease in DPOAE, and 12 ears (27%) lost DPOAE. Loss of DPOAE occurred in one ear (2%) at 0 years of age and four ears (9%) at 1 year of age. The time courses of DPOAEs in patients with OTOF mutations were divided into those with early loss and those with no change, indicating that the mechanism for deterioration of DPOAEs includes not only the OTOF mutations but also other common modifier factors. Most, but not all, AN patients who used hearing aids showed deterioration of DPOAEs after the start of using hearing aids. A few AN patients also showed deterioration of DPOAEs before using hearing aids. The adult AN group comprised 2 patients with OPA1 mutations, 2 with OTOF mutations, and 5 with indefinite causes. Four ears (22%) showed no change in DPOAE, 13 ears (72%) showed a decrease, and one ear (6%) showed a loss of DPOAE. Although the ratio of DPOAE decrease was higher in the adult AN group than in the pediatric AN group, the ratio of DPOAE loss was lower in the adult AN group. DPOAE was not lost in all four ears with OPA1 mutations and in all four ears with OTOF mutations in the adult group. CONCLUSIONS: DPOAE was decreased or lost in approximately 70% of pediatric and about 80% of adult AN patients. Eleven percent of pediatric AN patients lost DPOAEs by 1 year of age. Genetic factors were thought to have influenced the time course of DPOAEs in the pediatric AN group. In most adult AN patients, DPOAE was rarely lost regardless of the genetic cause.

Two-Point Method for Measuring the Temporal Modulation Transfer Function.

OBJECTIVE: The temporal modulation transfer function (TMTF) has been proposed to estimate the temporal resolution abilities of listeners with normal hearing and listeners with hearing loss. The TMTF data of patients would be useful for clinical diagnosis and for adjusting the hearing instruments at clinical and fitting sites. However, practical application is precluded by the long measurement time of the conventional method, which requires several measurement points. This article presents a new method to measure the TMTF that requires only two measurement points. DESIGN: Experiments were performed to estimate the TMTF of normal listeners and listeners with hearing loss to demonstrate that the two-point method can estimate the TMTF parameter and the conventional method. Sixteen normal hearing and 21 subjects with hearing loss participated, and the difference between the estimated TMTF parameters and measurement time were compared. RESULTS: The TMTF parameters (the peak sensitivity Lps and cutoff frequency fcutoff) estimated by the conventional and two-point methods showed significantly high correlations: the correlation coefficient for Lps was 0.91 (t(45) = 14.3; p < 10) and that for fcutoff was 0.89 (t(45) = 13.2; p < 10). There were no fixed and proportional biases. Therefore, the estimated values were in good agreement. Moreover, there was no systematic bias depending on the subject's profile. The measurement time of the two-point method was approximately 10 min, which is approximately one-third that of the conventional method. CONCLUSION: The two-point method enables the introduction of TMTF measurement in clinical diagnosis.

Pharmacological inhibition of cochlear mitochondrial respiratory chain induces secondary inflammation in the lateral wall: a potential therapeutic target for sensorineural hearing loss.

Cochlear lateral wall has recently been reported as a common site of inflammation, yet precise molecular mechanisms of the inflammatory responses remain elucidated. The present study examined the inflammatory responses in the lateral wall following acute mitochondrial dysfunction induced by a mitochondrial toxin, 3-nitropropionic acid (3-NP). Reverse-transcription (RT)-PCR revealed increases in the expression of the proinflammatory cytokines interleukin (IL)-1ß and IL-6. Immunohistochemistry showed an increase in the number of activated cochlear macrophages in the lateral wall, which were in close proximity to IL-6-expressing cells. A genome-wide DNA microarray analysis of the lateral wall revealed that 35% and 60% of the genes showing >2-fold upregulation at 1 d and 3 d post-3-NP administration, respectively, were inflammatory genes, including CC- and CXC-type chemokine genes. High expression of CCL-1, 2, and 3 at 1 d, and of CCL-1, 2, 3, 4, and 5, CCR-2 and 5, and CX3CR1 at 3 d post-3-NP administration, coupled with no change in the level of CX3CL1 expression suggested that macrophages and monocytes may be involved in the inflammatory response to 3-NP-mediated injury. Quantitative (q)RT-PCR showed a transient induction of IL-1ß and IL-6 expression within 24 h of 3-NP-mediated injury, followed by sustained expression of the chemoattractants, CCL-2, 4 and 5, up until 7 d after injury. The expression of CCL-2 and IL-6 was higher in animals showing permanent hearing impairment than in those showing temporary hearing impairment, suggesting that these inflammatory responses may be detrimental to hearing recovery. The present findings suggest that acute mitochondrial dysfunction induces secondary inflammatory responses in the lateral wall of the cochlear and that the IL-6/CCL-2 inflammatory pathway is involved in monocyte activation. Therefore, these secondary inflammatory responses may be a potential post-insult therapeutic target for treatments aimed at preventing the damage caused by acute mitochondrial dysfunction in the cochlear lateral wall.

Subgroups of enlarged vestibular aqueduct in relation to SLC26A4 mutations and hearing loss.

OBJECTIVES/HYPOTHESIS: To investigate possible association of hearing loss and SLC26A4 mutations with the subgroups of enlarged vestibular aqueduct (EVA) morphology in Japanese subjects with hearing loss. STUDY DESIGN: Retrospective multicenter study. METHODS: Forty-seven subjects who had vestibular aqueduct with midpoint diameter >1 mm by computed tomography of the temporal bone were enrolled at multiple sites across Japan, and DNA samples and clinical data were collected. EVA morphology was classified into four subgroups by the pattern of enlargement: aperture, aperture and midpoint, midpoint, and borderline enlargement. Venous blood DNA samples were subjected to polymerase chain reaction-based direct sequencing of all exons and exon-intron boundaries of the SLC26A4. RESULTS: Four novel SLC26A4 mutations were identified in the present study. SLC26A4 mutations were detected in almost all subjects with aperture, aperture and midpoint, and midpoint enlargement. In contrast, 71% of subjects with borderline enlargement had no SLC26A4 mutation. No significant difference was found in the distribution of truncating and nontruncating SLC26A4 mutations between the EVA subgroups. In addition, no significant correlation was observed between the EVA subgroups and hearing levels, incidence of hearing fluctuation, or progression of hearing loss. CONCLUSIONS: Subgroups of EVA morphology were significantly correlated with the presence or absence of SLC26A4 mutation. In a subgroup analysis of subjects with SLC26A4 mutations, however, differences in the EVA subgroups were not correlated with SLC26A4 genotypes or characteristics of hearing loss. LEVEL OF EVIDENCE: NA.

Age-related hearing loss and the factors determining continued usage of hearing aids among elderly community-dwelling residents.

While hearing aids are recommended for people with age-related hearing loss, many with impaired hearing do not use them. In this study, we investigated how many elderly people in the study area needed hearing aids, and the factors that determined continued wearing of the devices. The study area was Kurabuchi Town, Japan, where 1,437 residents (those aged 65 years or over) were eligible for participation in the study; 1,414 participated, of whom, 103 (7.3%) were already using hearing aids at the start of the study. After the primary screening, hearing aids were lent to 68 participants (4.8%) who did not already have one, 38 of whom (60.3% of the borrowers, representing 2.7% of the total aged population) went on to wear the hearing aid continuously. The Hearing Handicap Inventory for the Elderly (HHIE) score was significantly elevated among these 38 participants. This study indicated that hearing aids are of potential benefit to many local residents. Multivariate logistic regression revealed that HHIE scores were associated with the extent of HA usage. The adjusted odds ratio for a 1-unit increase in HHIE score was 1.08 (95% confidence interval: 1.02-1.14). Programs like this, in which people with impaired hearing are identified at the local level and given appropriate assistance, are useful models for future use in societies with aging populations.

Disease modifying therapies for Alzheimer's disease targeting Aß oligomers: implications for therapeutic mechanisms.

Several lines of evidence indicate that amyloid ß (Aß), particularly Aß oligomers (AßOs), plays a causative role in Alzheimer's disease. However, the mechanisms underlying the action of an anti-AßO antibody to clarify the toxic action of AßOs remain elusive. Here, we showed that the anti-AßO antibody (monoclonal 72D9) can modify the Aß aggregation pathway. We also found that 72D9 directly sequesters both extracellular and intraneuronal AßOs in a nontoxic state. Thus, therapeutic intervention targeting AßOs is a promising strategy for neuronal protection in Alzheimer's disease.

Sortilin is required for toxic action of Aß oligomers (AßOs): extracellular AßOs trigger apoptosis, and intraneuronal AßOs impair degradation pathways.

AIMS: We investigated the pathological relevance of the "Aß oligomer (AßO) cascade hypothesis" in 3xTg-AD mice. This study was also designed to elucidate the molecular mechanism underlying the toxic action of AßOs. MAIN METHODS: To target the extracellular AßOs in vivo, a monoclonal antibody specific for AßOs was developed using a novel method. Monoclonal 72D9 was intravenously administered to aged 3xTg-AD mice bearing the human AD pathology to investigate the relevance of the AßO cascade hypothesis. To further identify the AßO-binding molecule on the cell surface, small interfering RNA (siRNA) for sortilin was transfected into SH-SY5Y cells. The sortilin-dependent molecular mechanism underlying toxic action of AßOs and/or AßO endocytosis was also assessed in cultured cortical neurons forming synapses. KEY FINDINGS: The 72D9 immunotherapy of aged 3xTg-AD mice revealed that extracellular and intraneuronal AßOs are related, and that intraneuronal AßOs act upstream of tau. We also found that extracellular AßOs first act as a sortilin ligand, and then induce p75(NTF)-mediated apoptosis, endocytosis-induced attenuation of autophagy, or accumulation of AßOs in autophagosomes. SIGNIFICANCE: Taken together, these findings provide novel lines of evidence that sortilin governs the toxic action of extracellular AßOs, which affects the degradation and/or clearance of either intraneuronal AßOs or tau. Thus, therapeutic intervention targeting extracellular AßOs themselves or for preventing the interaction between intraneuronal AßOs and tau is a promising strategy to be developed for AD treatment.

Extracellular and intraneuronal HMW-AbetaOs represent a molecular basis of memory loss in Alzheimer's disease model mouse.

BACKGROUND: Several lines of evidence indicate that memory loss represents a synaptic failure caused by soluble amyloid ß (Aß) oligomers. However, the pathological relevance of Aß oligomers (AßOs) as the trigger of synaptic or neuronal degeneration, and the possible mechanism underlying the neurotoxic action of endogenous AßOs remain to be determined. RESULTS: To specifically target toxic AßOs in vivo, monoclonal antibodies (1A9 and 2C3) specific to them were generated using a novel design method. 1A9 and 2C3 specifically recognize soluble AßOs larger than 35-mers and pentamers on Blue native polyacrylamide gel electrophoresis, respectively. Biophysical and structural analysis by atomic force microscopy (AFM) revealed that neurotoxic 1A9 and 2C3 oligomeric conformers displayed non-fibrilar, relatively spherical structure. Of note, such AßOs were taken up by neuroblastoma (SH-SY5Y) cell, resulted in neuronal death. In humans, immunohistochemical analysis employing 1A9 or 2C3 revealed that 1A9 and 2C3 stain intraneuronal granules accumulated in the perikaryon of pyramidal neurons and some diffuse plaques. Fluoro Jade-B binding assay also revealed 1A9- or 2C3-stained neurons, indicating their impending degeneration. In a long-term low-dose prophylactic trial using active 1A9 or 2C3 antibody, we found that passive immunization protected a mouse model of Alzheimer's disease (AD) from memory deficits, synaptic degeneration, promotion of intraneuronal AßOs, and neuronal degeneration. Because the primary antitoxic action of 1A9 and 2C3 occurs outside neurons, our results suggest that extracellular AßOs initiate the AD toxic process and intraneuronal AßOs may worsen neuronal degeneration and memory loss. CONCLUSION: Now, we have evidence that HMW-AßOs are among the earliest manifestation of the AD toxic process in mice and humans. We are certain that our studies move us closer to our goal of finding a therapeutic target and/or confirming the relevance of our therapeutic strategy.

Repetitive transcranial magnetic stimulation (rTMS) for treatment of chronic tinnitus.

OBJECTIVE: There is compelling evidence that tinnitus is associated with functional alterations in the central nervous system. Repetitive transcranial magnetic stimulation (rTMS) is a potent tool for modifying neural activity at the stimulated area and at a distance along the functional anatomical connections. Depending on the stimulation parameters, cortical networks can be functionally disturbed or modulated in their activities. Low-frequency rTMS has been shown to result in a decrease in cortical excitability. The technique can alleviate tinnitus by modulating the excitability of neurons in the auditory cortex. We aimed to investigate the effects of low-frequency rTMS in patients and determine the factors that predict a beneficial outcome with rTMS treatment. METHODS: Sixteen patients (male 10, female 6) with chronic tinnitus underwent low-frequency (1Hz) rTMS (intensity: 110% motor threshold; number of stimuli: 1200) to the left auditory cortex. The treatment outcome was assessed with a visual analog scale (VAS) of loudness, annoyance and duration, loudness balance test, and tinnitus handicap inventory (THI). Therapeutic success was studied according to the patients' clinical characteristics. RESULTS: A significant reduction in the VAS (loudness and annoyance) occurred immediately after rTMS, with a gradual return to pretreatment levels after 7 days. The tinnitus patients with sudden deafness were significant resistant to rTMS treatment compared with those diagnosed with age-related hearing loss. CONCLUSION: These results support the potential of rTMS as a new therapeutic tool for the treatment of chronic tinnitus. Because this study was performed with a small sample size and showed high interindividual variability in treatment effects, further development of the technique is needed before it can be recommended for clinical applications.

Mesenchymal stem cell transplantation accelerates hearing recovery through the repair of injured cochlear fibrocytes.

Cochlear fibrocytes play important roles in normal hearing as well as in several types of sensorineural hearing loss attributable to inner ear homeostasis disorders. Recently, we developed a novel rat model of acute sensorineural hearing loss attributable to fibrocyte dysfunction induced by a mitochondrial toxin. In this model, we demonstrate active regeneration of the cochlear fibrocytes after severe focal apoptosis without any changes in the organ of Corti. To rescue the residual hearing loss, we transplanted mesenchymal stem cells into the lateral semicircular canal; a number of these stem cells were then detected in the injured area in the lateral wall. Rats with transplanted mesenchymal stem cells in the lateral wall demonstrated a significantly higher hearing recovery ratio than controls. The mesenchymal stem cells in the lateral wall also showed connexin 26 and connexin 30 immunostaining reminiscent of gap junctions between neighboring cells. These results indicate that reorganization of the cochlear fibrocytes leads to hearing recovery after acute sensorineural hearing loss in this model and suggest that mesenchymal stem cell transplantation into the inner ear may be a promising therapy for patients with sensorineural hearing loss attributable to degeneration of cochlear fibrocytes.

A minimal CENP-A core is required for nucleation and maintenance of a functional human centromere.

Chromatin clusters containing CENP-A, a histone H3 variant, are found in centromeres of multicellular eukaryotes. This study examines the ability of alpha-satellite (alphoid) DNA arrays in different lengths to nucleate CENP-A chromatin and form functional kinetochores de novo. Kinetochore assembly was followed by measuring human artificial chromosome formation in cultured human cells and by chromatin immunoprecipitation analysis. The results showed that both the length of alphoid DNA arrays and the density of CENP-B boxes had a strong impact on nucleation, spreading and/or maintenance of CENP-A chromatin, and formation of functional kinetochores. These effects are attributed to a change in the dynamic balance between assembly of chromatin containing trimethyl histone H3-K9 and chromatin containing CENP-A/C. The data presented here suggest that a functional minimum core stably maintained on 30-70 kb alphoid DNA arrays represents an epigenetic memory of centromeric chromatin.

Cholesterol granuloma surrounding the endolymphatic sac.

We report a unique case of cholesterol granuloma (CG) surrounding the endolymphatic sac (ES). A 49-year-old man presented with the left side of sensorineural hearing loss, tinnitus, and vertigo. Magnetic resonance and computed tomography imaging revealed a CG surrounding the left ES. The patient initially underwent left transmastoid surgical resection of the tumor. At the time of surgery, brown fluid was aspirated from the tumor, but no other tumors were found. Histopathological examination revealed that the tumor contained cholesterol crystals, confirming the diagnosis of CG. At his 12-month postoperative follow-up, there was no evidence of recurrence. We discuss the radiology, pathology, and surgical removal of CGs surrounding ES.

An artificially constructed de novo human chromosome behaves almost identically to its natural counterpart during metaphase and anaphase in living cells.

Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing alpha-satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input alpha-satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.

Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation.

Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. An investigation into the structural and the functional relationship in centromeric tandem repeats in HACs requires the ability to manipulate repeat substructure efficiently. We describe here a new method to rapidly amplify human alphoid tandem repeats of a few hundred base pairs into long DNA arrays up to 120 kb. The method includes rolling-circle amplification (RCA) of repeats in vitro and assembly of the RCA products by in vivo recombination in yeast. The synthetic arrays are competent in HAC formation when transformed into human cells. As short multimers can be easily modified before amplification, this new technique can identify repeat monomer regions critical for kinetochore seeding. The method may have more general application in elucidating the role of other tandem repeats in chromosome organization and dynamics.