A hearing was held in Riverhead for the 60-year-old suspect.
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Prosecutors in Suffolk County, New York, last month charged Heuermann with murdering Maureen Brainard-Barnes, who disappeared in 2007 while working as an escort, linking him to her death through DNA and other evidence.
Heuermann has already been charged with murdering three other escorts. Like Megan Waterman, Amber Costello and Melissa Barthelemy, the remains of Brainard-Barnes were found in a desolate spot along the ocean near Gilgo Beach.
Heuermann pleaded not guilty to the new second-degree murder charge. Defense attorney Michael Brown entered the not guilty plea on Heuermann's behalf. He was once again remanded to Suffolk County jail until his court date.
Brainard-Barnes is believed to be the first of the "Gilgo Four" to be killed. She was 25 years old at the time.
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Heuermann's now-estranged wife, Asa Ellerup, was also in court Tuesday.
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Prosecutors say she and his children were out of town when Brainard-Barnes disappeared and was killed, fitting an alleged pattern of Heuermann being home alone when the other three were killed.
The filing last month also revealed that prosecutors seized two phones from Heuermann at the time of his arrest that they said were held by him "in fictitious names and used for illicit activities." Prosecutors said Heuermann "utilized these phones in furtherance of hundreds of contacts with sex workers between 2020 and 2023."
Prosecutors additionally revealed they seized hundreds of electronic devices from Heuermann's Massapequa Park home and Manhattan office following his arrest.
Heuermann used the devices to search for the deceased victims and their family members; the status of the investigation; for software that would assist in wiping or erasing data from computers and other similar digital devices and purchase digital masking and forensic wiping tools, prosecutors said.
"Defendant's devices also contained the following: A collection of violent, bondage, and torture pornography preceding, during, and subsequent to the disappearances and murders of the aforementioned victims between 2007 and 2010; and prostitution-related searches preceding, during, and subsequent to the disappearances and murders of the aforementioned victims between 2007 and 2010."
The filing back in January also outlines the lengths prosecutors went to obtain DNA from Heuermann's family, including tracking his daughter on a Long Island Railroad train drinking from a gold-colored "Monster Java" can.
Investigators saw her toss the can into the trash. They recovered it and took it for analysis, prosecutors said.
Last summer, a special task force zeroed in on Heuermann, an architect who investigators linked to the women through DNA evidence, including a sample taken from pizza crust in the trash outside his Manhattan office.
Heuermann is a father of two from Massapequa Park -- just a few miles north of the dumping ground off Long Island's south shore.
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There are six other Gilgo Beach victims whose deaths remain unsolved.
The family of Brainard-Barnes said they have one final wish.
"I want to ask everyone to please remember the victims, Maureen, Megan, Melissa and Amber," Cann said. "I hope that everyone will also remember the other victims from whom charges have not been filed against any suspect."
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